ST6Gal-I Regulates Macrophage Apoptosis via α2-6 Sialylation of the TNFR1 Death Receptor

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 alpha 2-6 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 alpha 2-6 sialylation of selected receptors. To prevent loss of alpha 2-6 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 TNF alpha, which then activates TNFR1, we next evaluated the alpha 2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated alpha 2-6 sialylation, and this was associated with diminished TNF alpha-stimulated apoptosis. Correspondingly, removal of alpha 2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNF alpha-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated over-expressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated alpha 2-6 sialylation, and these cells were significantly protected against TNF alpha-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 alpha 2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNF alpha-stimulated apoptosis.