April 2012
Volume 53, Issue 4
Free
Retinal Cell Biology  |   April 2012
Glucosamine Modulates TNF-α–Induced ICAM-1 Expression and Function Through O-Linked and N-Linked Glycosylation in Human Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Ching-Long Chen
    From the Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China;
    Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
  • Chang-Min Liang
    From the Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China;
    Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
    Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China.
  • Yi-Hao Chen
    From the Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China;
    Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
  • Ming-Cheng Tai
    Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
  • Da-Wen Lu
    Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
  • Jiann-Torng Chen
    From the Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China;
    Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
  • Corresponding author: Jiann-Torng Chen, Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, 325 Cheng-Kung Road, Section 2, Taipei 114, Taiwan, Republic of China; jt66chen@ms32.hinet.net
Investigative Ophthalmology & Visual Science April 2012, Vol.53, 2281-2291. doi:10.1167/iovs.11-9291
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      Ching-Long Chen, Chang-Min Liang, Yi-Hao Chen, Ming-Cheng Tai, Da-Wen Lu, Jiann-Torng Chen; Glucosamine Modulates TNF-α–Induced ICAM-1 Expression and Function Through O-Linked and N-Linked Glycosylation in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(4):2281-2291. doi: 10.1167/iovs.11-9291.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose: The purpose of this article was to investigate the effects of glucosamine (GlcN) on the TNF-α–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-α–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 κB (NF-κB) signaling pathway was examined using an NF-κB 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-α–induced ICAM-1 expression and NF-κB activity in ARPE-19 cells. Alloxan increased ICAM-1 expression and NF-κB activity in TNF-α–induced ARPE-19 cells. GlcN and tunicamycin reduced the molecular mass of TNF-α–induced ICAM-1 in ARPE-19 cells. The proteasome inhibitor MG-132 suppressed the GlcN-induced reduction in the molecular mass of TNF-α–induced ICAM-1. GlcN also attenuated the adhesion activity of TNF-α–induced ICAM-1.

Conclusions: GlcN inhibits ICAM-1 expression and functions by modulating the O-linked glycosylation of factors involved in NF-κB signaling and by reducing the N-linked glycosylation of TNF-α–induced ICAM-1 in ARPE-19 cells. These effects may contribute to the GlcN-mediated anti-inflammatory effects in the eye.

Introduction
Glycoproteins can contain both N-linked (Asn-linked) and O-linked (Ser/Thr-linked) glycans of variable lengths and compositions. Some N-linked glycans exert a great influence on the conformation of the receptor proteins to which the glycoproteins are linked by modulating their ligand-binding activity. 1,2 O-linked glycosylation, a process similar to phosphorylation, is a common posttranslational modification of nuclear and cytosolic proteins. 
Intercellular adhesion molecule 1 (ICAM-1, CD54) is a type I transmembrane glycoprotein comprising five Ig superfamily (IgSF) domains, a transmembrane segment, and a cytoplasmic tail. 3 Most IgSF domains have N-linked glycans, which regulate both the conformation and function of ICAM-1. 4 ICAM-1 is synthesized in the cytoplasm and is then transferred to the cell membrane, where it mediates cell–cell and cell–matrix adhesive interactions involved in the immune functions. 5 Altering the N-linked glycosylation of ICAM-1 influences its biological functions. 4,6,7 Proinflammatory cytokines, such as TNF-α, can induce the expression of cell adhesion molecules, such as ICAM-1, in RPE cells. 8  
RPE cells are located between the neuroretina and the choroid coat. These cells comprise the outer blood–retinal barrier, participate in the selective transport of metabolites between the neuroretina and the choriocapillaries, phagocytose the outer segments shed from photoreceptors, and act as antigen-presenting cells, thereby participating in the immunogenic process. 911 ICAM-1 can be detected in the RPE cells of patients with posterior uveitis, and increased levels of ICAM-1 are thought to promote the extravasation of inflammatory cells into the retina. 12  
Glucosamine (GlcN), a naturally occurring amino monosaccharide, exerts some immunosuppressive effects in vitro and in vivo, and is used widely as an alternative therapeutic regimen for rheumatoid arthritis and osteoarthritis. 1315 It inhibits nuclear factor κB (NF-κB) activity and IL-1β bioactivity in rat chondrocytes by increasing the expression of the type II IL-1 decoy receptor, which suppresses the activation of T lymphoblasts and dendritic cells and prolongs cardiac allograft survival in vivo. 13,14 In our previous study, GlcN inhibited the expression of the ICAM-1 gene induced by TNF-α or IFN-γ in human RPE cells. 8 In a rat model of endotoxin-induced uveitis (EIU), we have demonstrated that treatment with GlcN inhibited experimental uveitis by blocking the NF-κB–dependent signaling pathway and ICAM-1 expression. 16  
GlcN is known to mimic the effects of the activation of the hexosamine biosynthesis pathway through the modulation of ganglioside levels, regulating mesangial cell growth and hypertrophy. 17 GlcN is also metabolized via the hexosamine biosynthesis pathway, which leads to the synthesis of uridine diphosphate N-acetylglucosamine, a substrate of multiple glycosylation reactions catalyzed by various GlcNAc transferases, including the unique O-linked GlcNAc (O-GlcNAc) transferase (OGT). 18,19 In a recent study, GlcN treatment produced results similar to those of OGT overexpression, such as reducing Inhibitor of κB (IκB) phosphorylation, NF-κB DNA-binding activity, and TNF-α and IL-6 mRNA expression, suggesting that GlcN can protect cells from stress by the O-linked glycosylation of relevant proteins. 20  
In the context of N-glycosylation, GlcN inhibits the biosynthesis and processing of N-linked oligosaccharides and causes marked and reversible changes in the nature of the lipid-linked oligosaccharides of glycoproteins. 21 The number of complex N-glycans and their degree of branching cooperate to regulate cell proliferation and differentiation via the metabolic flux through the hexosamine pathway. 22,23 We recently demonstrated that GlcN effectively suppresses the proliferation of RPE cells in vitro and that this effect appears to be mediated by the modification of N-glycans on the epidermal growth factor receptor (EGFR). 24 Jang et al. 25 showed that GlcN inhibits cyclo-oxygenase 2 (COX2) activity by preventing its cotranslational N-glycosylation; however, the effects of GlcN on O-linked and N-linked protein glycosylation, by which it affects the expression and function of TNF-α–induced ICAM-1 in ARPE-19 cells, is not fully understood. These mechanisms might clarify an important property of GlcN, whereby it reduces ICAM-1–mediated inflammatory responses in the eye. Therefore, the findings discussed previously prompted us to investigate the effects of GlcN on O-linked protein glycosylation and to ask whether N-linked glycosylation inhibits TNF-α–induced ICAM-1 in ARPE-19 cells. The objective of this study was to investigate whether GlcN can inhibit the expression and function of TNF-α–induced ICAM-1 by modulating its O-linked and N-linked glycosylation in ARPE-19 cells. 
Materials and Methods
RPE Cells
The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (Manassas, VA). The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM)–F-12 (Invitrogen–Gibco, Grand Island, NY) supplemented with 4 mM l-glutamine, 10% fetal bovine serum (FBS; Invitrogen–Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St Louis, MO) at 37°C under 5% CO2 in air. The culture medium was replaced twice weekly. All experiments were performed in serum-free medium unless otherwise stated. 
Western Blot Analysis
Confluent cultured cells were preincubated with or without 2.5 mM alloxan (an inhibitor of OGT that reduces O-GlcNAc protein levels) 26 for 1 hour, 100 μM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc, an inhibitor of N-acetylglucosaminidase [O-GlcNAcase, OGA] that increases O-GlcNAc protein levels) 27,28 for 1 hour, 2.5 μM MG-132 for 1 hour, or 2 μg/mL tunicamycin for 18 hours. They were then further incubated with or without 5 mM GlcN for 1 hour before stimulation with TNF-α (20 ng/mL) for 24 hours at 37°C. To measure ICAM-1 and O-GlcNAc, the cells were washed twice with PBS and detached by scraping. The cells were pelleted at 1000g, resuspended, and sonicated in cold lysis buffer (50 mM Tris-HCl [pH 7.5] 2% SDS, and 1 mM phenylmethylsulfonyl fluoride). The insoluble debris was removed by centrifugation at 16,000g at 4°C for 10 minutes. The protein content was determined using the bicinchoninic acid method (Pierce, Rockford, IL) with BSA as the standard. The lysates (20 μg) were resolved with one-dimensional SDS-PAGE (10% polyacrylamide gels). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA) that had been blocked with 5% (wt/vol) milk for 1 hour at room temperature, and then incubated overnight at 4°C with antibodies directed against ICAM-1 (diluted 1:1000 in Tris-buffered saline containing Tween-20 (TBST, 0.1% at 1X); Santa Cruz Biotechnology, Santa Cruz, CA), O-GlcNAc (CTD110.6; diluted 1:2000 in TBST; Covance, Berkeley, CA), OGT (diluted 1:1000 in TBST; Santa Cruz Biotechnology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; diluted 1:25,000 in TBST; Santa Cruz Biotechnology), and Na+/K+ ATPase (diluted 1:1000 in TBST; Santa Cruz Biotechnology). The membranes were washed and incubated with a horseradish-peroxidase-conjugated secondary antibody (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature, and the protein was visualized with an enhanced chemiluminescence procedure (enhanced chemiluminescence reagent; Millipore, Billerica, MA). The mean protein levels were measured densitometrically with Image J 1.46a (National Institutes of Health, Bethesda, MD). 
Flow Cytometry
Flow cytometry was used to assess the expression of ICAM-1 in ARPE-19 cells in response to TNF-α treatment. Confluent cultured cells were preincubated with or without 2.5 mM alloxan or 100 μM PUGNAc for 1 hour and further incubated with or without 5 mM GlcN for 1 hour before they were stimulated with TNF-α (20 ng/mL) for 24 hours at 37°C. A corresponding culture was left untreated at each time point. After treatment with TNF-α, the cells were harvested with PBS-based enzyme-free dissociation buffer (Invitrogen–Gibco): a membrane-filtered, isotonic and enzyme-free aqueous formulation of salts, chelating agents (EDTA), and cell-conditioning agents in Ca2+- and Mg2+-free PBS. The cells were washed in PBS and incubated in 0.5% normal goat serum and 0.5% BSA in PBS for 30 minutes to block the nonspecific binding sites. To determine their ICAM-1 expression, 106 cells/10 μL cells were incubated with primary antibody (FITC-labeled mouse anti-human ICAM-1; BioSource, Camarillo, CA) at 4°C for 1 hour. The cells were washed in PBS and analyzed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA). The isotype control was FITC-labeled mouse isotype IgG1 (BD Pharmingen, San Diego, CA). Ten thousand cells were analyzed for each sample. 
OGT Overexpression
pcDNA3.1–OGT and pcDNA3.1 were purchased from Open Biosystems (Huntsville, AL) and Invitrogen Life Technologies (Carlsbad, CA), respectively. ARPE-19 cells were transfected with the pcDNA3.1 and pcDNA3.1-OGT vectors according to the manufacturers' protocols. After incubation for 24 hours, the transfected cells were harvested. The expression of OGT was confirmed by Western blot analysis. 
Fractionation Experiment
Fractionation was performed with a commercially available kit (Plasma Membrane Protein Extraction Kit; BioVision, Mountain View, CA), according to the manufacturer's instructions. In brief, confluent cultured cells were preincubated with or without 2.5 mM alloxan or 100 μM PUGNAc for 1 hour and further incubated with or without 5 mM glucosamine for 1 hour before they were stimulated with TNF-α (20 ng/mL) for 24 hours at 37°C. The cells were scraped in PBS, pelleted (600g for 5 minutes), and then washed once with 1 mL of ice-cold PBS. The cells were resuspended in 1 mL of homogenizer buffer mix in an ice-cold Dounce homogenizer. The homogenized cells were incubated on ice 30 to 50 times. The homogenate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 700g for 10 minutes at 4°C; the supernatant was collected and the pellet was discarded. The supernatant was transferred to a new vial and centrifuged at 10,000g for 30 minutes at 4°C. The supernatant was the cytosolic fraction and the pellet contained the total cellular membrane proteins (from both the plasma membrane and the cellular organellar membranes). The cytosolic and plasma membrane fractions were each resolved by SDS-PAGE and transferred to PVDF membranes for the Western blotting analysis of ICAM-1, GAPDH, and Na+/K+ ATPase. 
NF-κB Reporter Assay
ARPE-19 cells (3 × 104/well) were plated and maintained in DMEM/F-12 with 10% FBS in 24-well dishes for 24 hours. To measure their NF-κB activity, the ARPE-19 cells were cotransfected with pCMV-luciferase (Promega, Milan, Italy) and either pTAL–secreted alkaline phosphatase (SEAP) or pNF-κB–SEAP (Clontech, San Jose, CA) at a ratio of 1:4 for 4 hours using the polycationic detergent Lipofectamine Plus (Invitrogen–Gibco), according to the manufacturer's instructions. The ARPE-19 cells were maintained for 20 hours and then preincubated with or without 2.5 mM alloxan or 100 μM PUGNAc for 1 hour and further incubated with or without 5 mM GlcN for 1 hour before they were stimulated with TNF-α (20 ng/mL) for 24 hours at 37°C. The experiments for each treatment were performed in triplicate. The SEAP activity was determined in the culture supernatants, and the luciferase activity was measured in the cell lysates to normalize the transfection efficiency. The luciferase activity was assessed with the Promega Dual-Luciferase Reporter 1000 Assay System. 
ICAM-1 Adhesion Assay
THP-1 cells were grown in RPMI-1640 medium containing 10% FBS in a culture flask. ARPE-19 cells were seeded in 24-well plates. Only confluent monolayers were preincubated with or without 2.5 mM alloxan or 100 μM PUGNAc for 1 hour and then further incubated with or without 5 mM GlcN for 1 hour before they were stimulated with TNF-α (20 ng/mL) for 24 hours at 37°C. The THP-1 cells were labeled for 30 minutes with 5 μmol/L calcein-AM (Molecular Probes, Inc., Eugene, OR) and 5.0 × 105 cells were then cocultured with the ARPE-19 cells for 1 hour. The cocultured cells were washed three times with PBS. Fluorescent images were obtained at 485 nm excitation and 538 nm emission with a SPOT II fluorescence microscope equipped with a digital camera, with the SPOT II data acquisition software (Diagnostic Instruments, Sterling Heights, MI). To quantify cell adhesion, the calcein-AM fluorescence intensity was measured at 485 nm excitation and 538 nm emission with a Fluoroskan ELISA plate reader (Labsystems Oy, Helsinki, Finland). 
Statistical Analysis
Normally distributed continuous variables were compared with ANOVA. When a significant difference between the groups was apparent, multiple comparisons of their means were made with Tukey's procedure. Data are presented as means ± SEM. Each result is representative of at least three independent experiments. All statistical assessments were two-sided and evaluated at the 0.05 level of significance. Statistical analyses were performed with the SPSS 19.0 statistical software (SPSS Inc., Chicago, IL). 
Results
GlcN-Induced Increase in O-GlcNAc Protein Levels in ARPE-19 Cells
To evaluate the effects of GlcN on O-GlcNAc protein levels in ARPE-19 cells, we treated ARPE-19 cells with TNF-α with or without GlcN, alloxan, or PUGNAc. As shown in Figure 1, GlcN dose-dependently increased the O-GlcNAc protein levels in the TNF-α-treated ARPE-19 cells, as detected by Western blotting. A comparison of the effects of GlcN, alloxan, and PUGNAc on the O-GlcNAc protein levels in TNF-α-treated ARPE-19 cells showed that GlcN reversed the effect of alloxan. In contrast, there was no obvious difference between the effects of PUGNAc with and without GlcN. Overall, these results suggest that coincubation with GlcN and alloxan reduced the O-GlcNAc protein levels, thus exerting the opposite effect to alloxan treatment alone. Coincubation with GlcN and PUGNAc had no synergistic effect, however, and did not increase the O-GlcNAc protein levels compared with PUGNAc treatment alone. 
Figure 1.
 
Effect of GlcN on O-GlcNAc protein levels in ARPE-19 cells. ARPE-19 cells were pretreated with GlcN (2.5 mM, 5 mM), alloxan (2.5 mM), PUGNAc (100 μM), or GlcN with either alloxan or PUGNAc, for 1 hour. The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against O-GlcNAc and GAPDH. Mean protein O-GlcNAc levels were measured with densitometric analysis and normalized to GAPDH. The data are means ± SEM of three independent experiments. The differences in the protein O-GlcNAc levels in ARPE-19 cells of the different groups were compared with ANOVA; Tukey's test was used for the post hoc analysis. ns, not significant; ***P < 0.001; *P < 0.05; ### P < 0.001 versus the TNF-α group.
Figure 1.
 
Effect of GlcN on O-GlcNAc protein levels in ARPE-19 cells. ARPE-19 cells were pretreated with GlcN (2.5 mM, 5 mM), alloxan (2.5 mM), PUGNAc (100 μM), or GlcN with either alloxan or PUGNAc, for 1 hour. The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against O-GlcNAc and GAPDH. Mean protein O-GlcNAc levels were measured with densitometric analysis and normalized to GAPDH. The data are means ± SEM of three independent experiments. The differences in the protein O-GlcNAc levels in ARPE-19 cells of the different groups were compared with ANOVA; Tukey's test was used for the post hoc analysis. ns, not significant; ***P < 0.001; *P < 0.05; ### P < 0.001 versus the TNF-α group.
GlcN-Induced Reduction in TNF-α–Induced ICAM-1 Expression in ARPE-19 Cells via the Increase in O-GlcNAc Protein Levels
We investigated whether treatment with GlcN to increase the O-GlcNAc protein levels influenced TNF-α–induced ICAM-1 expression. To evaluate the effects of O-linked glycosylation on TNF-α–induced ICAM-1 expression, we used Western blotting to measure the ICAM-1 protein levels in ARPE-19 cells treated with or without GlcN, alloxan, or PUGNAc. 
Without TNF-α stimulation, neither the control, GlcN, alloxan (or PUGNAc), nor GlcN and alloxan (or PUGNAc) together affected the expression or molecular mass of ICAM-1 protein (Fig. 2A, Fig. 2B, lanes 1–4). As shown in Figure 2A, after stimulation with TNF-α, the ICAM-1 protein levels increased (lane 5) compared with the control levels (lane 1). GlcN reduced the expression of TNF-α–induced ICAM-1 (lane 5 versus lane 6). GlcN treatment induced the expression of a smaller ICAM-1, with an apparent molecular mass of approximately 60 to 75 kDa (strong accumulation of a distinct ICAM-1 of approximately 65 kDa; lane 6 versus lane 8) compared with the normally expressed approximately 85 kDa protein (lane 1). Alloxan increased the expression of TNF-α–induced ICAM-1 without influencing its molecular mass (lane 5 versus lane 7). In cells coincubated with GlcN and alloxan, the expression of TNF-α–induced ICAM-1 protein decreased compared with that in cells treated with alloxan alone (lane 7 versus lane 8). Interestingly, low-molecular-mass TNF-α–induced ICAM-1 proteins were produced by GlcN and alloxan together, but not by alloxan treatment alone (lane 7 versus lane 8). As shown in Figure 2B, however, GlcN reduced the expression and molecular mass of TNF-α–induced ICAM-1 (lane 5 versus lane 6). PUGNAc reduced the expression of TNF-α–induced ICAM-1 without influencing its molecular mass (lane 5 versus lane 7). The expression of normal-molecular-mass TNF-α–induced ICAM-1 protein was reduced and the low-molecular-mass TNF-α–induced ICAM-1 protein was increased by GlcN and PUGNAc together compared with the protein produced after PUGNAc treatment alone (lane 7 versus lane 8). GlcN treatment alone caused a complete loss of normal-molecular-weight ICAM-1, so the addition of PUGNAc had no further effect (lanes 6–8). 
Figure 2.
 
Effects of GlcN treatment on TNF-α–induced ICAM-1 expression and O-GlcNAc protein levels in ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; **P < 0.01 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; and ns, not significant versus the TNF-α group. (B) Comparison of the effects of GlcN and PUGNAc on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. ns, not significant; ***P < 0.001 versus the TNF-α group; ### P < 0.001. (C) Comparison of the effects of GlcN, PUGNAc, and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 2.
 
Effects of GlcN treatment on TNF-α–induced ICAM-1 expression and O-GlcNAc protein levels in ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; **P < 0.01 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; and ns, not significant versus the TNF-α group. (B) Comparison of the effects of GlcN and PUGNAc on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. ns, not significant; ***P < 0.001 versus the TNF-α group; ### P < 0.001. (C) Comparison of the effects of GlcN, PUGNAc, and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
We used flow cytometry to examine the cell-surface expression of ICAM-1 protein after TNF-α stimulation with or without GlcN, alloxan, or PUGNAc. Consistent with the results of the Western blotting analysis, GlcN, PUGNAc, or both reduced the expression of TNF-α–induced ICAM-1. Alloxan increased the expression of TNF-α–induced ICAM-1, but this effect was reduced by GlcN treatment (Fig. 2C). Taken together, these results demonstrate that GlcN reduces TNF-α–induced ICAM-1 expression by increasing the O-GlcNAc protein levels in ARPE-19 cells. 
Relationship between the Attenuating Effects of GlcN on the TNF-α–Induced Activation of the NF-κB Pathway in ARPE-19 Cells and Increased O-GlcNAc Protein Levels
As described previously, GlcN reduced TNF-α–induced ICAM-1 expression by increasing O-GlcNAc levels in ARPE-19 cells (Figs. 1 and 2). Our previous study showed that GlcN inhibits ICAM-1 expression and synthesis by inhibiting NF-κB activity in TNF-α–treated ARPE-19 cells. 8 This prompted us to investigate whether GlcN attenuates the TNF-α–induced activation of the NF-κB pathway by increasing O-GlcNAc levels in ARPE-19 cells. We used an NF-κB reporter assay to study whether GlcN attenuates NF-κB activity by increasing O-linked protein glycosylation in TNF-α–treated ARPE-19 cells. As shown in Figure 3, GlcN inhibited NF-κB reporter activity in TNF-α–treated ARPE-19 cells. Alloxan increased NF-κB reporter activity in TNF-α–treated ARPE-19 cells, but this activity was blocked by GlcN. PUGNAc inhibited NF-κB reporter activity in TNF-α–induced ARPE-19 cells, and this activity was augmented by GlcN. Taken together, these results suggest that GlcN attenuates NF-κB signaling by increasing O-GlcNAc protein levels in TNF-α–treated ARPE-19 cells. 
Figure 3.
 
Effects of GlcN on the TNF-α–induced activation of the NF-κB pathway in ARPE-19 cells via its increase of O-GlcNAc protein levels. ARPE-19 cells were pretreated with GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. An NF-κB reporter assay was used. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 3.
 
Effects of GlcN on the TNF-α–induced activation of the NF-κB pathway in ARPE-19 cells via its increase of O-GlcNAc protein levels. ARPE-19 cells were pretreated with GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. An NF-κB reporter assay was used. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Effects of GlcN on the Molecular Mass of TNF-α–Induced ICAM-1 Expressed in ARPE-19 Cells and Lack of a Relationship with Increased O-GlcNAc Protein Levels
Previous studies have shown that GlcN treatment mimics the effects of OGT overexpression, which increases O-GlcNAc levels and is associated with the attenuation of the lipopolysaccharide (LPS)-induced increase in ICAM-1 expression in neonatal rat ventricular myocytes (NRVMs). 20 We compared the effects of GlcN treatment and OGT overexpression on TNF-α–induced ICAM-1 expression in ARPE-19 cells. Figure 4 shows that the overexpression of the OGT gene was associated with reduced TNF-α–induced ICAM-1 levels. This effect is consistent with that induced by GlcN treatment. Interestingly, the reduced molecular mass of ICAM-1 was achieved only with the GlcN treatment (lane 5 versus lane 6). Incubation of OGT-overexpressing cells with GlcN reduced both the expression and the molecular mass of TNF-α–induced ICAM-1 protein in ARPE-19 cells (lane 5 versus lane 8). These data suggest that the GlcN-induced reduction in the molecular mass of TNF-α–induced ICAM-1 in ARPE-19 cells is unrelated to the increase in O-GlcNAc protein levels. 
Figure 4.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to increased O-GlcNAc protein levels. Comparison of the effects of GlcN and OGT overexpression on the molecular mass and expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were transfected with pcDNA3.1–OGT and/or pretreated with GlcN (5 mM). The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, or were transfected with pcDNA3.1–OGT. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against ICAM-1 and GAPDH.
Figure 4.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to increased O-GlcNAc protein levels. Comparison of the effects of GlcN and OGT overexpression on the molecular mass and expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were transfected with pcDNA3.1–OGT and/or pretreated with GlcN (5 mM). The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, or were transfected with pcDNA3.1–OGT. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against ICAM-1 and GAPDH.
Relationship Between the Effect of GlcN on the Molecular Mass of TNF-α–Induced ICAM-1 Expressed in ARPE-19 Cells and the N-Glycosylation of ICAM-1
We have reported that GlcN significantly reduced the N-glycosylation of EGFR. 24 N-glycan biosynthesis can be blocked with pharmacological tools, such as tunicamycin, which inhibits the formation of a lipid-linked oligosaccharide precursor. 29 Tunicamycin is an inhibitor of protein N-glycosylation, which inhibits ICAM-1 N-glycosylation, leading to the expression of glycosylated ICAM-1 with a molecular mass of 50 to 95 kDa. 30 Therefore, tunicamycin was used as a positive control to evaluate the effect of GlcN treatment on ICAM-1 N-glycosylation. We also evaluated whether GlcN modulates the N-linked glycosylation of TNF-α–induced ICAM-1 and whether this reduces its molecular mass in ARPE-19 cells. As shown in Figure 5A, tunicamycin alone reduced the molecular mass of ICAM-1 (∼55 kDa) but did not affect ICAM-1 expression in TNF-α–induced ARPE-19 cells (lane 5 versus lane 7). Compared with tunicamycin, GlcN reduced both the molecular mass (∼65 kDa) and expression of TNF-α–induced ICAM-1 in ARPE-19 cells (lane 5 versus lane 6). GlcN also reduced the expression of TNF-α–induced ICAM-1 in tunicamycin-treated ARPE-19 cells (lane 7 versus lane 8). Moreover, GlcN and tunicamycin did not reduce the molecular mass of ICAM-1 in ARPE-19 cells in the absence of TNF-α stimulation (lanes 1–4). This suggests that GlcN and tunicamycin modulate the N-glycosylation of newly synthesized ICAM-1. These results show that GlcN and tunicamycin, inhibitors of the biosynthesis and processing of N-linked oligosaccharides, reduce the molecular mass of nascent ICAM-1. The different molecular mass of the nascent ICAM-1 suggests that its degree of N-glycosylation differs after treatment with GlcN or tunicamycin. 
Figure 5.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to the reduced N-glycosylation of ICAM-1. (A) Comparison of the effects of GlcN and tunicamycin on the molecular mass of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, tunicamycin, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, tunicamycin, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH. (B) Suppressive effect of the proteasome inhibitor MG-132 on GlcN-induced N-glycosylation in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, MG-132, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, MG-132, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH.
Figure 5.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to the reduced N-glycosylation of ICAM-1. (A) Comparison of the effects of GlcN and tunicamycin on the molecular mass of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, tunicamycin, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, tunicamycin, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH. (B) Suppressive effect of the proteasome inhibitor MG-132 on GlcN-induced N-glycosylation in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, MG-132, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, MG-132, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH.
Abolition of the Suppressive Effect of GlcN on ICAM-1 N-Glycosylation by the Proteasome Inhibitor MG-132
A recent study reported that the proteasome inhibitor MG-132 inhibits the GlcN-stimulated expression of hypoglycosylated COX2 in IL-1β-treated A549 cells when given as GlcN-HCl at a concentration of 1 mM, but not when given at a concentration of 5 mM. 25 This prompted us to investigate whether the mechanism underlying the modulation of N-linked glycosylation of TNF-α–induced ICAM-1 by GlcN is associated with proteasome-mediated proteolytic mechanisms. As shown in Figure 5B, MG-132 slightly inhibited the TNF-α–induced expression of ICAM-1 protein (lane 5 versus lane 7) and MG-132 abolished the inhibitory effects of GlcN on the N-glycosylation of TNF-α–induced ICAM-1 (lane 6 versus lane 8). These results suggest that the proteasomal pathway plays a role in the production of TNF-α–induced hypoglycosylated (low-molecular-mass) ICAM-1 in GlcN-treated ARPE-19 cells. 
Effect of GlcN on the Modulation of the Membrane and Cytosolic Fractions of ICAM-1 in TNF-α–Treated ARPE-19 Cells by Preventing ICAM-1 N-Glycosylation
As described previously, GlcN inhibited the N-glycosylation of TNF-α–induced ICAM-1 (Figs. 5A, 5B, lane 6), with the concomitant production of hypoglycosylated forms of ICAM-1. Therefore, we asked whether the nascent hypoglycosylated forms of TNF-α–induced ICAM-1 are transferred to the cell surface or remain in the cytoplasm in ARPE-19 cells. As shown in Figure 6A, GlcN reduced ICAM-1 expression and induced the production of the low-molecular-mass form in the membrane and cytosolic fractions (lane 5 versus lane 6). Alloxan increased ICAM-1 expression in the membrane and cytosolic fractions (lane 5 versus lane 7). Alloxan and GlcN cotreatment increased the expression of low-molecular-mass ICAM-1 in the membrane and cytosolic fractions but did not increase the expression of normal-molecular-mass ICAM-1 (lane 6 versus lane 8). 
Figure 6.
 
Effects of GlcN on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase. (B) Comparison of the effects of GlcN and PUGNAc on ICAM-1 in the membrane and cytosolic fractions of TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase.
Figure 6.
 
Effects of GlcN on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase. (B) Comparison of the effects of GlcN and PUGNAc on ICAM-1 in the membrane and cytosolic fractions of TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase.
PUGNAc reduced ICAM-1 expression in the membrane and cytosolic fractions (Fig. 6B, lane 5 versus lane 7). PUGNAc and GlcN cotreatment reduced the expression of low-molecular-mass ICAM-1 in the membrane and cytosolic fractions (lane 6 versus lane 8). GlcN, PUGNAc, and both combined reduced ICAM-1 expression in the membrane and cytosolic fractions. These results suggest that decreasing O-GlcNAc levels with alloxan or increasing O-GlcNAc levels with GlcN or PUGNAc influenced ICAM-1 expression on the cell membrane and in the cytosol. Intriguingly, GlcN induced the formation of nascent hypoglycosylated ICAM-1, which could have been transferred from the cytoplasm to the cell membrane, by preventing N-glycosylation in TNF-α–treated ARPE-19 cells. 
GlcN Blocks the Adhesion Activity of ARPE-19 Cells via TNF-α–Induced ICAM-1
We next asked whether low-molecular-mass ICAM-1 on the cell surface displays normal adhesion activity in ARPE-19 cells. GlcN inhibited the adhesion activity of TNF-α–induced ICAM-1 in ARPE-19 cells (Fig. 7). Alloxan slightly improved the adhesion activity of ICAM-1 in TNF-α–treated ARPE-19 cells, but GlcN inhibited this improvement. PUGNAc slightly inhibited the adhesion activity in TNF-α–treated ARPE-19 cells, and GlcN suppressed this activity further. These results suggest that the modulation of O-linked glycosylation by GlcN, alloxan, or PUGNAc interferes with ICAM-1 expression, which then influences the adhesion activity of ICAM-1 in TNF-α–treated ARPE-19 cells. GlcN could have reduced ICAM-1 expression by increasing O-linked glycosylation and could have blocked ICAM-1 adhesion activity by reducing the N-glycosylation of ICAM-1, however. Taken together, these results suggest that ICAM-1 adhesion activity is influenced by both its N-glycosylation and its TNF-α–induced expression in ARPE-19 cells. 
Figure 7.
 
Effect of GlcN on the adhesion activity of ICAM-1 in TNF-α–induced ARPE-19 cells by reducing its N-linked glycosylation. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. An ICAM-1 adhesion assay was performed. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 7.
 
Effect of GlcN on the adhesion activity of ICAM-1 in TNF-α–induced ARPE-19 cells by reducing its N-linked glycosylation. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. An ICAM-1 adhesion assay was performed. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Discussion
In a previous study, we reported that GlcN effectively inhibits ICAM-1 expression and synthesis by inhibiting NF-κB activity in human RPE cells stimulated with TNF-α 8 ; however, the mechanism by which GlcN affects the expression and adhesion activity of ICAM-1 was unclear. In this study, we further demonstrated the effects of GlcN and the mechanism underlying its effect on the expression and adhesion activity of ICAM-1 in response to the inflammatory cytokine TNF-α. We report for the first time that GlcN increases O-GlcNAc protein levels, attenuates NF-κB reporter activity, and inhibits the expression of ICAM-1 in TNF-α–treated ARPE-19 cells. We also found that GlcN reduces the N-glycosylation of ICAM-1, which reduces its molecular mass and blocks its adhesion activity in TNF-α–treated ARPE-19 cells. The effects of GlcN on the N-glycosylation and expression of ICAM-1 played an important role in influencing the adhesion activity of ICAM-1 in TNF-α–induced ARPE-19 cells. 
GlcN treatment increases O-GlcNAc levels in many tissues. 31 Consistent with the literature discussed previously, 31 we showed here that GlcN treatment increased O-GlcNAc protein levels in ARPE-19 cells. O-GlcNAc protein levels are regulated by the activities of two key enzymes: OGT, which catalyzes the addition of O-GlcNAc, 32,33 and OGA, a neutral hexosaminidase responsible for O-GlcNAc removal. 34 Alloxan, an inhibitor of OGT, reduces O-GlcNAc protein levels, whereas PUGNAc, an inhibitor of OGA and responsible for the removal of O-GlcNAc from proteins, increases O-GlcNAc protein levels. 2628 In the present study, GlcN treatment reversed the effects of alloxan on O-GlcNAc levels and had a similar effect to that of PUGNAc on O-GlcNAc levels in TNF-α–induced ARPE-19 cells. 
Rajapakse et al. 35 demonstrated that GlcN exerts its anti-inflammatory effects by increasing O-GlcNAc protein levels and reducing TNF-α–induced ICAM-1 expression in human umbilical vein endothelial cells. GlcN treatment also increased O-GlcNAc protein levels in NRVMs, and this effect was associated with the attenuation of the LPS-induced increase in NF-κB signaling, intracellular TNF-α, and ICAM-1 expression. 20 Other authors have shown that the levels of several proinflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) are elevated during LPS-induced EIU. 36 In our previous studies, GlcN suppressed exogenous TNF-α–induced ICAM-1 expression in ARPE-19 cells, LPS-induced NF-κB signaling, and ICAM-1 expression in EIU. 8,16 Consistent with these studies, 8,16,20,36 ICAM-1 expression was reduced in TNF-α–induced ARPE-19 cells treated with GlcN in our study, and this effect was similar to that of PUGNAc. In contrast, alloxan increased ICAM-1 expression. We also found that GlcN inhibited the increase in ICAM-1 expression induced by alloxan and reduced ICAM-1 expression when given with PUGNAc to TNF-α–induced ARPE-19 cells. Our flow-cytometry results show that treatment with GlcN, alloxan, or PUGNAc affected the expression of ICAM-1 in TNF-α–induced ARPE-19 cells, which is consistent with our Western blot analysis. As we know, GlcN and PUGNAc, both of which increase O-GlcNAc levels but by different mechanisms, inhibit acute inflammatory and neointimal responses to endoluminal arterial injury 37 ; however, GlcN treatment can affect the hexosamine pathway, influencing ganglioside levels and N-glycans other than O-GlcNAc. 17,22,23 PUGNAc also inhibits β-hexosamindases other than OGA. 38,39 Therefore, both GlcN and PUGNAc could alter the processing of glycoconjugates in addition to O-GlcNAc. We also examined whether OGT overexpression mimics the effects of GlcN in attenuating TNF-α–induced ICAM-1 expression in ARPE-19 cells. Our results suggest that OGT overexpression attenuates TNF-α–induced ICAM-1 expression in ARPE-19 cells, as does GlcN. Taken together, these results support the notion that GlcN treatment reduces ICAM-1 expression in TNF-α–induced ARPE-19 cells and that this effect is associated with an increase in O-GlcNAc proteins. These findings are consistent with the results of other studies. 20,35  
The transcription factor NF-κB acts as a critical regulator of cytokine production, lymphocyte activation, and cell proliferation. 40,41 The activation of NF-κB requires posttranslational modifications, including its phosphorylation, acetylation, and glycosylation. Treatment with TNF-α or other activating agents stimulates IκB kinase, which phosphorylates IκB and thereby induces its degradation. 42 This leads to the dissociation and translocation of NF-κB into the nucleus and the activation of its target genes. 40,43 High glucose and GlcN levels and OGT overexpression increase the O-linked glycosylation of the NF-κB p65 subunit and the human VCAM1 promoter activity in mesangial cells. 44 A previous study, however, demonstrated that increasing the O-GlcNAc protein modification of NF-κB p65 with GlcN and PUGNAc treatments inhibits TNF-α–induced inflammatory responses in isolated rat aortic smooth muscle cells by inhibiting the phosphorylation of NF-κB p65 and promoting the binding of IκBα to NF-κB p65. 45 A reciprocal relationship between O-linked glycosylation and phosphorylation has been described for the NF-κB p65 subunit, suggesting that O-linked glycosylation and phosphorylation may modulate one another. 45 Our previous study also demonstrated that GlcN inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with TNF-α by blocking the NF-κB subunit p65. 8 Consistent with these data, we have shown in this study that NF-κB reporter activity was inhibited by GlcN, PUGNAc, or both, and was increased by alloxan, but was blocked by GlcN in TNF-α–induced ARPE-19 cells. 
We also found that the pretreatment of TNF-α–induced ARPE-19 cells with GlcN led to the expression of an ICAM-1 protein with a molecular mass lower than that of the normally expressed protein. This was also observed in cells coincubated with GlcN and alloxan, GlcN and PUGNAc, or GlcN under OGT-overexpressing conditions, but not in cells treated with alloxan or PUGNAc, or under OGT-overexpressing conditions alone. These findings suggest that the unique effect of GlcN in inducing the expression of the lower-molecular-mass ICAM-1 in TNF-α–treated ARPE-19 cells, which was not observed with PUGNAc treatment or OGT overexpression, is unrelated to the capacity of GlcN to increase O-GlcNAc protein levels. 
ICAM-1 is a glycoprotein with a molecular mass of 80 to 114 kDa, depending on the degree of its N-glycosylation. 46 The inhibition of N-glycosylation by GlcN alters the molecular mass of glucose transporter 1 in L6 myocytes, lipoprotein apoB-100 in HepG2 cells, and COX2 in A540 cells. 25,47,48 GlcN also inhibits the N-linked glycosylation of EGFR, which inhibits RPE proliferation. 24 Our observation that GlcN treatment altered the molecular mass of ICAM-1 from 60 to 75 kDa (∼65 kDa) in TNF-α–treated ARPE-19 cells is consistent with these previously reported data. Tunicamycin treatment alone shifted the molecular mass of ICAM-1 to approximately 55 kDa. With GlcN and tunicamycin cotreatment, the molecular mass of ICAM-1 shifted from approximately 65 kDa to approximately 55 kDa. We found that treatment with tunicamycin induced the formation of more hypoglycosylated ICAM-1 than did GlcN treatment. Furthermore, the expression of the low-molecular-mass species of ICAM-1 decreased after treatment with GlcN or GlcN and tunicamycin compared with their expression after tunicamycin treatment alone, in TNF-α–treated ARPE-19 cells. These results suggest that GlcN reduces the molecular mass of ICAM-1 by reducing its N-glycosylation. 
N-linked protein glycosylation occurs in the endoplasmic reticulum and reflects the folding status of the glycoproteins passing through the endoplasmic reticulum, which provides a protein quality-control system. Folding-defective polypeptides or components that are not incorporated into protein complexes must eventually be cleared by endoplasmic reticulum–associated degradation involving the ubiquitin–proteasome proteolytic process. 49,50 MG-132, an inhibitor of proteasomal proteolysis, inhibits the production of hypoglycosylated COX2 induced by GlcN in IL-1β–treated A549 cells. 25 Consistent with the results of that study, 25 we found in this study that MG-132 blocked the synthesis of the new low-molecular-mass ICAM-1 produced in response to the treatment of TNF-α–induced ARPE-19 cells with GlcN. Interestingly, MG-132, at a concentration of 20 μM, has been shown to inhibit ICAM-1 expression by blocking the NF-κB–dependent signal transduction pathway in TNF-α–induced human RPE cells. 51 We used a much lower concentration of MG-132 (2.5 μM) and found that MG-132 at this concentration slightly inhibited the TNF-α–induced expression of ICAM-1 protein but more strongly inhibited the GlcN-mediated expression of hypoglycosylated ICAM-1. Collectively, these findings suggest that low-dose MG-132 partially blocks the GlcN-mediated expression of hypoglycosylated ICAM-1 but not the TNF-α–induced expression of ICAM-1 protein. 
Glycosylation affects the stability, activity, and/or cellular location of proteins. The translation of these glycoproteins occurs in the cytoplasm and cotranslational N-glycosylation occurs in the endoplasmic reticulum. The glycosylated membrane protein is then transported to the plasma membrane. In the present study, GlcN simultaneously inhibited the expression of the membrane and cytosolic fractions of ICAM-1 through O-linked protein glycosylation in TNF-α–induced ARPE-19 cells. We also found that the nascent low-molecular-mass ICAM-1 could be transferred to the cell surface; however, a defect in the N-glycosylation of ICAM-1 protein influences its biological function, including its conformation and immune-related functions. 3,6,7 GlcN reduces the activity of COX2 by preventing the cotranslational N-glycosylation of the enzyme. 25 Our previous study showed that GlcN inhibits the N-glycosylation of EGFR and inhibits EGF-induced RPE proliferation. 24 Consistent with earlier studies, 3,6,7,24,25 we found that GlcN reduces the N-glycosylation of ICAM-1, which plays an important role in its adhesion activity, in TNF-α–induced ARPE-19 cells. 
In conclusion, GlcN effectively inhibited ICAM-1 expression and synthesis by inhibiting NF-κB activity in TNF-α–induced ARPE-19 cells. This inhibition of ICAM-1 expression was associated with increased O-GlcNAc protein levels. The appearance of low-molecular-mass ICAM-1 and the inhibition of its adhesion activity were associated with the N-glycosylation of ICAM-1 in TNF-α–induced ARPE-19 cells. Our findings suggest a possible mechanism underlying the purported anti-inflammatory effects of GlcN in ocular inflammatory disorders. 
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Footnotes
 Supported in part by Grant TSGH-C100-008-006-6-S01 from the Tri-Service General Hospital and Grant NSC-99-2314-B-016-009-MY3 from the National Science Council.
Footnotes
 Disclosure: C.L. Chen, None; C.M. Liang, None; Y.H. Chen, None; M.C. Tai, None; D.W. Lu, None; J.T. Chen, None
Figure 1.
 
Effect of GlcN on O-GlcNAc protein levels in ARPE-19 cells. ARPE-19 cells were pretreated with GlcN (2.5 mM, 5 mM), alloxan (2.5 mM), PUGNAc (100 μM), or GlcN with either alloxan or PUGNAc, for 1 hour. The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against O-GlcNAc and GAPDH. Mean protein O-GlcNAc levels were measured with densitometric analysis and normalized to GAPDH. The data are means ± SEM of three independent experiments. The differences in the protein O-GlcNAc levels in ARPE-19 cells of the different groups were compared with ANOVA; Tukey's test was used for the post hoc analysis. ns, not significant; ***P < 0.001; *P < 0.05; ### P < 0.001 versus the TNF-α group.
Figure 1.
 
Effect of GlcN on O-GlcNAc protein levels in ARPE-19 cells. ARPE-19 cells were pretreated with GlcN (2.5 mM, 5 mM), alloxan (2.5 mM), PUGNAc (100 μM), or GlcN with either alloxan or PUGNAc, for 1 hour. The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against O-GlcNAc and GAPDH. Mean protein O-GlcNAc levels were measured with densitometric analysis and normalized to GAPDH. The data are means ± SEM of three independent experiments. The differences in the protein O-GlcNAc levels in ARPE-19 cells of the different groups were compared with ANOVA; Tukey's test was used for the post hoc analysis. ns, not significant; ***P < 0.001; *P < 0.05; ### P < 0.001 versus the TNF-α group.
Figure 2.
 
Effects of GlcN treatment on TNF-α–induced ICAM-1 expression and O-GlcNAc protein levels in ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; **P < 0.01 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; and ns, not significant versus the TNF-α group. (B) Comparison of the effects of GlcN and PUGNAc on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. ns, not significant; ***P < 0.001 versus the TNF-α group; ### P < 0.001. (C) Comparison of the effects of GlcN, PUGNAc, and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 2.
 
Effects of GlcN treatment on TNF-α–induced ICAM-1 expression and O-GlcNAc protein levels in ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or GlcN with either alloxan or PUGNAc. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; **P < 0.01 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; and ns, not significant versus the TNF-α group. (B) Comparison of the effects of GlcN and PUGNAc on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or GlcN with either alloxan or PUGNAc. ns, not significant; ***P < 0.001 versus the TNF-α group; ### P < 0.001. (C) Comparison of the effects of GlcN, PUGNAc, and alloxan on the expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 3.
 
Effects of GlcN on the TNF-α–induced activation of the NF-κB pathway in ARPE-19 cells via its increase of O-GlcNAc protein levels. ARPE-19 cells were pretreated with GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. An NF-κB reporter assay was used. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 3.
 
Effects of GlcN on the TNF-α–induced activation of the NF-κB pathway in ARPE-19 cells via its increase of O-GlcNAc protein levels. ARPE-19 cells were pretreated with GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, alloxan, or GlcN with either alloxan or PUGNAc. An NF-κB reporter assay was used. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 4.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to increased O-GlcNAc protein levels. Comparison of the effects of GlcN and OGT overexpression on the molecular mass and expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were transfected with pcDNA3.1–OGT and/or pretreated with GlcN (5 mM). The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, or were transfected with pcDNA3.1–OGT. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against ICAM-1 and GAPDH.
Figure 4.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to increased O-GlcNAc protein levels. Comparison of the effects of GlcN and OGT overexpression on the molecular mass and expression of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were transfected with pcDNA3.1–OGT and/or pretreated with GlcN (5 mM). The cells were then treated with TNF-α (20 ng/mL) for 24 hours in the absence or presence of GlcN, or were transfected with pcDNA3.1–OGT. Whole-cell lysates were prepared and analyzed with immunoblotting using antibodies directed against ICAM-1 and GAPDH.
Figure 5.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to the reduced N-glycosylation of ICAM-1. (A) Comparison of the effects of GlcN and tunicamycin on the molecular mass of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, tunicamycin, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, tunicamycin, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH. (B) Suppressive effect of the proteasome inhibitor MG-132 on GlcN-induced N-glycosylation in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, MG-132, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, MG-132, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH.
Figure 5.
 
Effects of GlcN on the molecular mass of TNF-α–induced ICAM-1 expressed in ARPE-19 cells and its relationship to the reduced N-glycosylation of ICAM-1. (A) Comparison of the effects of GlcN and tunicamycin on the molecular mass of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, tunicamycin, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, tunicamycin, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH. (B) Suppressive effect of the proteasome inhibitor MG-132 on GlcN-induced N-glycosylation in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, MG-132, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, MG-132, or both. Whole-cell lysates were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1 and GAPDH.
Figure 6.
 
Effects of GlcN on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase. (B) Comparison of the effects of GlcN and PUGNAc on ICAM-1 in the membrane and cytosolic fractions of TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase.
Figure 6.
 
Effects of GlcN on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. (A) Comparison of the effects of GlcN and alloxan on the membrane and cytosolic fractions of ICAM-1 in TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, alloxan, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase. (B) Comparison of the effects of GlcN and PUGNAc on ICAM-1 in the membrane and cytosolic fractions of TNF-α–induced ARPE-19 cells. ARPE-19 cells were pretreated with GlcN, PUGNAc, or both. The cells were then treated with TNF-α in the absence or presence of GlcN, PUGNAc, or both. A plasma membrane protein extraction kit was used. The membrane and cytosolic fractions of ICAM-1 protein were prepared and analyzed by immunoblotting with antibodies directed against ICAM-1, GAPDH, and Na+/K+ ATPase.
Figure 7.
 
Effect of GlcN on the adhesion activity of ICAM-1 in TNF-α–induced ARPE-19 cells by reducing its N-linked glycosylation. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. An ICAM-1 adhesion assay was performed. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
Figure 7.
 
Effect of GlcN on the adhesion activity of ICAM-1 in TNF-α–induced ARPE-19 cells by reducing its N-linked glycosylation. ARPE-19 cells were pretreated with GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. The cells were then treated with TNF-α in the absence or presence of GlcN, alloxan, PUGNAc, or GlcN with either alloxan or PUGNAc. An ICAM-1 adhesion assay was performed. The results are the means ± SEM of three independent experiments. ***P < 0.001 versus the TNF-α group; ### P < 0.001 versus the TNF-α + alloxan group; +++ P < 0.001 versus the TNF-α + PUGNAc group.
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