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. 2011 Nov 11;286(45):39654-62.
doi: 10.1074/jbc.M111.276063. Epub 2011 Sep 19.

ST6Gal-I regulates macrophage apoptosis via α2-6 sialylation of the TNFR1 death receptor

Zhongyu Liu  1 , Amanda F Swindall, Robert A Kesterson, Trenton R Schoeb, Daniel C Bullard, Susan L Bellis
Affiliations

ST6Gal-I regulates macrophage apoptosis via α2-6 sialylation of the TNFR1 death receptor

Zhongyu Liu et al. J Biol Chem. .

Abstract

Macrophages play a central role in innate immunity, however mechanisms regulating macrophage survival are not fully understood. Herein we describe a novel apoptotic pathway involving α2-6 sialylation of the TNFR1 death receptor by the ST6Gal-I sialyltransferase. Variant glycosylation of TNFR1 has not previously been implicated in TNFR1 function, and little is known regarding the TNFR1 glycan composition. To study sialylation in macrophages, we treated U937 monocytic cells with PMA, which stimulates both macrophage differentiation and apoptosis. Interestingly, macrophage differentiation induces ST6Gal-I down-regulation, leading to reduced α2-6 sialylation of selected receptors. To prevent loss of α2-6 sialylation, we forced constitutive expression of ST6Gal-I, and found that this strongly inhibited PMA-induced apoptosis. Given that PMA-mediated apoptosis is thought to result from up-regulation of TNFα, which then activates TNFR1, we next evaluated the α2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated α2-6 sialylation, and this was associated with diminished TNFα-stimulated apoptosis. Correspondingly, removal of α2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNFα-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated overexpressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated α2-6 sialylation, and these cells were significantly protected against TNFα-stimulated apoptosis. Moreover, greater numbers of thioglycollate-induced peritoneal cells were observed in transgenic mice. These collective results highlight a new mechanism of TNFR1 regulation, and further intimate that loss of α2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNFα-stimulated apoptosis.

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Figures

FIGURE 1.
FIGURE 1.
ST6Gal-I inhibits PMA-induced suppression of cell growth. A, Western blot shows ST6Gal-I expression in control or PMA-treated Par, EV, or ST6Gal-I transduced (ST6) U937 cells. B, cells were suspended in media with PMA or without PMA (control, C), and then seeded into culture dishes at a density of 2 × 106 (dashed line). Cell number was quantified 24 h following this incubation. Values represent means and S.E. from three independent experiments.
FIGURE 2.
FIGURE 2.
ST6Gal-I protects U937 cells from PMA-induced apoptosis. A, cells were treated with or without PMA (control, C) for 24 h and then stained for the apoptotic markers, activated caspases 3 and 7 (orange). Nuclei were counterstained with Hoechst (blue). B, percentage of apoptotic cells was quantified by counting cells positive for activated caspases 3 and 7 relative to total cells from multiple microscopic fields. C, cells treated with or without PMA were stained with FITC annexin V and propidium iodide (PI). Lower right quadrant, annexin V positive; Upper right quadrant, annexin V + PI positive. D, quantification of annexin-positive cells (representing combined totals from lower and upper-right quadrants). Values represent means and S.E. from three independent experiments.
FIGURE 3.
FIGURE 3.
PMA-induced down-regulation of ST6Gal-I is associated with loss of α2-6 sialylation from TNFR1. A, cells were treated with or without PMA for 24 h, and then the amount of TNFα secreted into culture media was measured by ELISA. Data are plotted as fold TNFα induced by PMA as compared with control samples. Values represent means and S.E. from three independent experiments. B, lysates from cells treated with or without PMA were incubated with agarose-conjugated SNA lectin. α2-6 sialylated proteins were precipitated and blotted for TNFR1. C, total TNFR1 levels were detected by blots of whole cell lysates. D, cell lysates were treated with neuraminidase at 37 °C for 1 h, and then Western blot was performed with anti-TNFR1 antibody.
FIGURE 4.
FIGURE 4.
N-glycans are the major source of α2-6 sialic acids on TNFR1. A, cell lysates were treated with or without PNGaseF, and then TNFR1 immunoblots were performed. B, cell lysates were treated with or without PNGaseF, and then lysates were incubated with agarose-conjugated SNA lectin. α2-6 sialylated proteins were precipitated and blotted for TNFR1.
FIGURE 5.
FIGURE 5.
Decreased α2-6 sialylation of TNFR1 promotes U937 cell apoptosis. A, cells were treated with or without TNFα plus CHX for 24 h (C, control). Cells were then analyzed by flow cytometry for annexin V and PI. Values represent means and S.E. for three independent experiments. B, annexin V and PI reactivity were analyzed in Par cells treated with neuraminidase at 37 °C for 1 h, followed by treatment with TNFα plus CHX for 24 h. C, Par cells were transduced with two different shRNA sequences (#1 and #2) against ST6Gal-I. Western blotting was performed to confirm knockdown. D, Par, EV, and shRNA-transduced cells were treated with TNFα plus CHX for 24 h, and then evaluated for annexin V and PI reactivity. E, lysates from Par, EV, ST6, or shRNA (sequence #2) cells were incubated with MAA-agarose and α2-3 sialylated proteins were precipitated by centrifugation. Precipitates were immunoblotted for TNFR1.
FIGURE 6.
FIGURE 6.
ST6Gal-I activity causes increased α2-6 sialylation of TNFR1 in vivo. A, construct used to generate ST6Gal-I-expressing transgenic mice. B, splenic homogenates were prepared from wild-type (WT) or transgenic mice, and immunoblotting was performed with anti-V5 antibody and anti-ST6Gal-I antibody. C, peritoneal cells collected 96 h following thioglycollate injection were immunoblotted for ST6Gal-I. D, α2-6 sialylated proteins from peritoneal cell lysates were precipitated with SNA-agarose and immunoblotted for TNFR1. E, cell lysates from wild-type and transgenic mice were immunoblotted for TNFR1.
FIGURE 7.
FIGURE 7.
ST6Gal-I inhibits apoptosis of macrophages from ST6Gal-I transgenic mice. A, peritoneal cells were harvested at 96 h following thioglycollate injection and nucleated cell counts were obtained using a hemacytometer (n = 10 for each group of animals). B, peritoneal cells were stained with annexin V and PI, and the apoptosis rate was analyzed by flow cytometry (n = 11 WT; n = 10 line 1; n = 9 line 2). C and D, peritoneal cells were cultured overnight, and the adherent fraction (representing macrophages) was treated with TNFα plus CHX for 24 h. Annexin V and PI staining revealed significantly lower levels of apoptosis in each of the two transgenic lines. (For panel C, n = 5 for each group; for panel D, n = 8 for each group.)
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