Activation of caspase-3 and degradation of Nanog are induced in cells overexpressing <i>3OST-5</i>.

<p> (A) Viability of cells overexpressing <i>3OST-5</i>. Viability was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043440#s2" target="_blank">Materials and Methods</a>. The values shown are the means ± SD of triplicate measurements from one representative experiment after normalization against control cells (arbitrary value = 1). (B) Measurement of the rate of apoptosis in mESCs using an annexin V-FITC kit at 2 days after transfection with the <i>3OST-5</i> expression construct. The values shown are the means ± SD after normalization against control cells (arbitrary value = 1). (C) FACS analysis, using the HS4C3 antibody and annexin V, of mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct (<i>left graph</i>) or of mESCs cultured for 8 days in the absence of LIF (−LIF) (<i>right graph</i>). The histograms show the ratio of the HS4C3 and annexin V double positive cells to control cells (<i>left graph</i>) or mESCs cultured in the presence of LIF (+LIF) (<i>right graph</i>) (value = 1). Values are the means ± SD. (D) and (E) Western blot analysis, using antibodies against cleaved caspase-3 or Nanog, of mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct (D, <i>left two lanes</i> and E) or of mESCs cultured for 8 days in the absence of LIF (−LIF) (D, <i>right two lanes</i>). The histograms show mean densitometric readings ± SD after normalization against control cells (D, <i>left two lanes</i> and E), or mESCs cultured in the presence of LIF (+LIF) (D, <i>right two lanes</i>) (arbitrary value = 1). (F) Real time PCR analysis of <i>Nanog</i> in mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct. The values shown are means ± SD after normalization against control cells (arbitrary value = 1). <i>*</i>, <i>P</i><0.01; <i>**</i>, <i>P</i><0.05. Three independent experiments were performed in each case.</p

The expression of Fas increased on the surface of cells overexpressing <i>3OST-5</i>.

<p>(A) FACS analysis of cell surface Fas (<i>left panel)</i> or total Fas (<i>right panel</i>) in cells overexpressing <i>3OST-5</i> using an anti-Fas antibody (<i>black line</i>, control cells; <i>red line</i>, cells overexpressing <i>3OST-5</i>). The <i>gray line</i> shows the result obtained from cells not treated with primary antibody. As described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043440#s2" target="_blank">Materials and Methods</a>, mESCs were permeabilized to observe total Fas expression or not permeabilized to observe Fas expression on the cell surface. (B) FACS analysis, using an anti-Fas antibody, of Fas on the surface of cells that were overexpressing <i>3OST-5</i> and had been treated with brefeldin A. The histograms show the ratio of the mean fluorescence intensity of the treated cells to that of non-treated control cells (arbitrary value = 1). Values are the means ± SD. (C) FACS analysis of cell surface Fas in mESCs cultured for 8 days in the absence of LIF using an anti-Fas antibody. In the <i>left panel</i>, a histogram shows a representative result of the FACS analysis (<i>black line</i>, in the presence of LIF; <i>red line</i>, in the absence of LIF). The <i>gray line</i> shows the result obtained from cells not treated with primary antibody. In the <i>right panel</i>, the values shown are the mean fluorescence intensity ± SD after normalization against mESCs cultured in the presence of LIF (arbitrary value = 1). *, <i>P</i><0.01; **, <i>P</i><0.05. Three independent experiments were performed in each case.</p

Subcellular localization of HS4C3-binding epitope and Fas, and scheme for Fas-mediated differentiation via HS4C3-binding epitope.

<p>(A) Immunostaining, using HS4C3 and anti-Fas antibodies, of permeabilized mESCs in the presence of LIF (+LIF) or mESCs cultured for 5 or 7 days in the absence of LIF (−LIF day 5 or day 7). Scale bar, 5 µm. Representative confocal images are shown. (B) Scheme for the induction of differentiation by the activation of Fas signaling via the HS4C3-binding epitope. (1) The HS4C3-binding epitope is synthesized on HSPGs in the Golgi. (2) The HS4C3-binding epitope on HSPGs interacts with Fas in the Golgi. (3) Fas then translocates into lipid rafts on the cell surface through interaction between the HS4C3-binding epitope and Fas. Fas signaling is activated by this redistribution of Fas from the Golgi into the lipid rafts. (4) Fas signaling activates caspase-3, and then activated caspase-3 degrades Nanog. (5) mESCs differentiate due to the degradation of Nanog. HSPGs, heparan sulfate proteoglycans.</p

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

Differentiation induced by overexpression of the HS4C3-binding epitope is inhibited by an inhibitor of Fas signaling.

<p>(A) and (B) Western blot analysis using antibodies against cleaved caspase-3 and Nanog in the presence or absence of IETD or DEVD, peptides that block caspase-8 and caspase-3, respectively. Representative results are shown. The histograms show mean densitometric readings ± SD after normalization against cells overexpressing <i>3OST-5</i> but not treated with IETD or DEVD (arbitrary value = 1). mESCs were analyzed at 2 days after transfection with the <i>3OST-5</i> expression construct. (C) Representative photomicrographs of transfected cells in the presence or absence of IETD. Scale bars, 200 µm. A triple asterisk (***) indicates a high magnification image of the boxed area (Scale bars, 100 µm). mESCs were analyzed at 4 days after transfection with the <i>3OST-5</i> expression construct. (D) Self-renewal assay with cells overexpressing <i>3OST-5</i> treated with IETD or DEVD. <i>Left panels</i> show photographs of representative colonies. Scale bars, 200 µm. The <i>right panel</i> shows the proportion of AP-positive colonies. The values shown are the mean ± SD. Two days after transfection, mESCs were replated in ESC medium with LIF. mESCs were cultured with inhibitors throughout the period from transfection to AP staining. (E) Real time PCR analysis of markers of the undifferentiated state. The values shown are means ± SD after normalization against cells overexpressing <i>3OST-5</i> but not treated with IETD (arbitrary value = 1). mESCs were analyzed at 4 days after transfection with the <i>3OST-5</i> expression construct. (F) Self-renewal assay after treatment with IETD in the presence or absence of LIF. The ratio of AP-positive colonies is shown. The values shown are the mean ± S.D. IETD, Ac-IETD-CHO; DEVD, Ac-DEVD-CHO; AP, alkaline phosphatase. *, <i>P</i><0.01; **, <i>P</i><0.05. Three independent experiments were performed in each case.</p

Fas signaling is activated by redistribution of Fas into lipid rafts in cells overexpressing <i>3OST-5</i>.

<p> (A) Western blot analysis of raft and non-raft fractions, using anti-Flotillin-1 (raft), anti-transferrin receptor (non-raft), and anti-Fas antibodies, of mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct (<i>upper two panels</i>) or mESCs at 6 days after LIF withdrawal (<i>lower two panels</i>). At least two independent experiments were performed. Representative results are shown. (B) Western blot analysis, using antibodies against uncleaved and cleaved caspase-8, of mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct (<i>left</i> and <i>middle right panels</i>) or mESCs at 8 days after LIF withdrawal (<i>middle left</i> and <i>right panels</i>). The histograms show mean densitometric readings ± SD after normalization against control cells (<i>left</i> and <i>middle right panels</i>) or mESCs cultured in the presence of LIF (<i>middle left</i> and <i>right panels</i>) (arbitrary value = 1). Three independent experiments were performed. <i>*</i>, <i>P</i><0.01; <i>**</i>, <i>P</i><0.05. (C) FACS analysis, using the anti-Fas antibody, of mESCs at 2 days after transfection with the <i>Fas</i> expression construct. In the <i>left panel</i>, a histogram shows a representative result of the FACS analysis (<i>black line</i>, control cells; <i>red line</i>, cells overexpressing <i>Fas</i>). The <i>gray line</i> shows the result obtained for cells that were not treated with the primary antibody. In the <i>right panel</i>, the values shown are means ± SD after normalization against control cells (arbitrary value = 1). Three independent experiments were performed. <i>*</i>, <i>P</i><0.01. (D) Self-renewal assay in cells overexpressing <i>Fas</i>. The proportion of AP-positive colonies is shown. The values shown are the mean ± SD. Two days after transfection, mESCs were replated in ESC medium with or without LIF. <i>*</i>, <i>P</i><0.01. (E) Mutations and truncations of the recombinant Fas ectodomain. (F) and (G) Overlay assay using the GST-fused recombinant Fas ectodomain. F-1 and G-1 show a western blot using the HS4C3 antibody. The single asterisk (*) shows the effect of the increase in the HS4C3-binding epitope on several core proteins in cells overexpressing <i>3OST-5</i>. F-2 and G-2,-4 show the overlay assay using the Fas ectodomain (F-2, aa 19–168) or fragments of the Fas ectodomain (G-2, aa 19–38; G-4, aa 39–168). F-3 and G-3,-5 show the overlay assay using the Fas ectodomain (F-3, aa 19–168) or fragments of the Fas ectodomain (G-3, aa 19–38; G-5, aa 39–168) pre-mixed with HS4C3 antibody. F-4 shows the overlay assay using the mutated Fas ectodomain (aa 19–168). The double asterisk (**) shows increased binding of the Fas ectodomain in cells overexpressing <i>3OST-5</i>. β-actin was used as a loading control for each sample (F-5 and G-6). mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct were used for each analysis. Two independent experiments were performed. Representative results are shown. GST, glutathione S-transferase.</p

Decrease in the HS4C3-binding epitope inhibits differentiation and apoptosis.

<p> (A) and (J) Real time PCR analysis of <i>3OST-5</i> in stable (A) and transient (J) <i>3OST-5</i> KD cells. The values shown are means ± SD after normalization against control cells (arbitrary value = 1). (B) FACS analysis using the anti-HS antibody HS4C3 (<i>black line</i>, control ESCs; <i>blue line,</i> stable <i>3OST-5-1</i> KD ESCs; <i>red line</i>, control EBs; <i>green line</i>, stable <i>3OST-5-1</i> KD EBs). The control cells were transfected with the construct that targeted <i>EGFP</i>. The <i>gray line</i> shows the result obtained for cells not treated with primary antibody. (C) Self-renewal assay with stable <i>3OST-5</i> KD cells. <i>Left panels</i> show photographs of representative colonies. Scale bars, 200 µm. The <i>right panel</i> shows the proportion of AP-positive colonies. The values shown are the mean ± SD. (D) and (E) Real time PCR analysis of marker genes in stable <i>3OST-5</i> KD cells at 6 days after LIF withdrawal (D) and at 3 (<i>Fgf5</i>, <i>Goosecoid</i>, and <i>Sox17</i>), 4 (<i>Nanog</i> and <i>Oct3/4</i>), or 12 (<i>Pax6</i>) days after EB formation (E). The values shown are means ± SD after normalization against control cells (arbitrary value = 1). (F) Western blot analysis, using anti-pErk1/2 and Erk1/2 antibodies, of cells stimulated with Fgf4. The histograms show mean densitometric readings ± SD expressed as the ratio p-Erk1/2/Erk1/2. Representative results are shown. (G) Real time PCR analysis of marker genes, <i>Fgf5</i> and <i>Goosecoid</i>, in cells treated with IETD at 3 days after EB formation. The values shown are means ± SD after normalization against nontreated cells (arbitrary value = 1). (H) FACS analysis using the anti-HS antibody HS4C3 in cells overexpressing <i>3OST-5</i> at 2 days after EB formation. The values shown are mean fluorescence intensity ± SD. (I) Real time PCR analysis of <i>Fgf5</i> and <i>Goosecoid</i> in cells overexpressing <i>3OST-5</i> at 0–4 days after EB formation (<i>black line</i>, control cells; <i>red line</i>, cells overexpressing <i>3OST-5</i>). The values shown are means ± SD from duplicate measurements from one representative experiment. (K) FACS analysis using the anti-HS antibody HS4C3 (<i>black line</i>, control cells; <i>red line</i>, transient <i>3OST-5-2</i> KD cells). The <i>gray line</i> shows the result obtained for cells not treated with primary antibody. (L) Measurement of apoptosis in transient <i>3OST-5</i> KD cells using an annexin V-FITC kit at 2 days after transfection. The values shown are the means ± SD after normalization against control cells in the absence of LIF (arbitrary value = 1). KD, knockdown; RNAi, RNA interference; EB, embryoid body. *, <i>P</i><0.01; **, <i>P</i><0.05. Three independent experiments were performed in each case.</p

Increase in the HS4C3-binding epitope induces differentiation.

<p>(A) FACS analysis, using the HS4C3 antibody, of mESCs after LIF withdrawal up to day 8. mESCs were cultured in the presence of LIF until day 0. In the <i>left panel</i>, a histogram shows a representative result of the FACS analysis (<i>black line</i>, in the presence of LIF; <i>red line</i>, 4 days after LIF withdrawal; <i>green line</i>, 6 days after LIF withdrawal; <i>blue line</i>, 8 days after LIF withdrawal). The <i>gray line</i> shows the result obtained from cells not treated with primary antibody. In the <i>right panel</i>, the values shown are the mean fluorescence intensity ± SD after normalization against mESCs cultured in the presence of LIF (arbitrary value = 1). (B) Immunostaining, using the HS4C3 antibody, of non-permeabilized mESCs cultured in the presence of LIF (+LIF) or mESCs cultured for 7 days in the absence of LIF (−LIF) (<i>upper panel</i>). <i>Lower panel</i> shows DIC images. Scale bar, 5 µm. Representative confocal images are shown. (C) Real time PCR analysis of mESCs after LIF withdrawal up to day 6. The values shown are means ± SD. (D) FACS analysis, using the HS4C3 antibody, of mESCs at 2 days after transfection with the <i>3OST-5</i> expression construct. In the <i>left panel</i>, a histogram shows a representative result of the FACS analysis (<i>black line</i>, control cells; <i>red line</i>, cells overexpressing <i>3OST-5</i>). The <i>gray line</i> shows the result obtained from cells not treated with primary antibody. The control cells were transfected with empty pCAGI vector. In the <i>right panel</i>, the values shown are the mean fluorescence intensity ± SD after normalization against control cells (arbitrary value = 1). (E) Self-renewal assay with cells overexpressing <i>3OST-5</i>. <i>Left panels</i> show photographs of representative colonies. A high magnification image is shown at the bottom of each photograph to the right. Scale bars, 200 µm. <i>Right panel</i> shows the proportion of AP-positive colonies. The values shown are the mean ± SD. Two days after transfection, mESCs were replated in ESC medium with or without LIF. (F) and (G) Real time PCR analysis of markers of the undifferentiated (F) and differentiated state (G) in mESCs at 4 days after transfection with the <i>3OST-5</i> expression construct. The values shown are means ± SD after normalization against control cells (arbitrary value = 1). DIC, Differential interference contrast. <i>*</i>, <i>P</i><0.01; <i>**</i>, <i>P</i><0.05. At least three independent experiments were performed in each case.</p