Glucosamine Modulates TNF-α-Induced ICAM-1 Expression and Function Through O-Linked and N-Linked Glycosylation in Human Retinal Pigment Epithelial Cells

PURPOSE. The purpose of this article was to investigate the effects of glucosamine (GlcN) on the TNF-alpha-induced expression of intercellular adhesion molecule 1 (ICAM-1) and the function of ICAM-1 in ARPE-19 cells in vitro. METHODS. We quantified protein levels of TNF-alpha-induced ICAM-1 in ARPE-19 cells with Western blotting. The effects of GlcN on O-linked glycosylation, and therefore on ICAM-1 expression, were compared after the addition of alloxan, an inhibitor of O-linked N-acetylglucosamine transferase (OGT), or O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase (O-GlcNAcase [OGA]), or after OGT gene overexpression. The effect of GlcN on the N-linked glycosylation of ICAM-1 was evaluated by the change in its molecular mass on Western blotting. The effect of O-linked glycosylation on the nuclear factor kappa B (NF-kappa B) signaling pathway was examined using an NF-kappa B reporter gene assay. The effect of GlcN on ICAM-1 adhesion activity was examined using an ICAM-1 adhesion assay. RESULTS. GlcN, PUGNAc, and OGT overexpression inhibited TNF-alpha-induced ICAM-1 expression and NF-kappa B activity in ARPE-19 cells. Alloxan increased ICAM-1 expression and NF-kappa B activity in TNF-alpha-induced ARPE-19 cells. GlcN and tunicamycin reduced the molecular mass of TNF-alpha-induced ICAM-1 in ARPE-19 cells. The proteasome inhibitor MG-132 suppressed the GlcN-induced reduction in the molecular mass of TNF-alpha-induced ICAM-1. GlcN also attenuated the adhesion activity of TNF-alpha-induced ICAM-1. CONCLUSIONS. GlcN inhibits ICAM-1 expression and functions by modulating the O-linked glycosylation of factors involved in NF-kappa B signaling and by reducing the N-linked glycosylation of TNF-alpha-induced ICAM-1 in ARPE-19 cells. These effects may contribute to the GlcN-mediated anti-inflammatory effects in the eye. (Invest Ophthalmol Vis Sci. 2012; 53: 2281-2291) DOI:10.1167/iovs.11-9291