Glucosamine Hydrochloride Specifically Inhibits COX-2 by Preventing COX-2 N-Glycosylation and by Increasing COX-2 Protein Turnover in a Proteasome-dependent Manner

Abstract

COX-2 and its products, including prostaglandin E2, are involved in many inflammatory processes. Glucosamine (GS) is an amino monosaccharide and has been widely used for alternative regimen of (osteo)arthritis. However, the mechanism of action of GS on COX-2 expression remains unclear. Here we describe a new action mechanism of glucosamine hydrochloride (GS-HCl) to tackle endogenous and agonistdriven COX-2 at protein level. GS-HCl (but not GS sulfate, N-acetyl GS, or galactosamine HCl) resulted in a shift in the molecular mass of COX-2 from 72–74 to 66–70 kDa and concomitant inhibition of prostaglandin E2 production in a concentration- dependent manner in interleukin (IL)-1 -treated A549 human lung epithelial cells. Remarkably, GS-HCl-mediated decrease in COX-2 molecular mass was associated with inhibition of COX-2 N-glycosylation during translation, as assessed by the effect of tunicamycin, the protein N-glycosylation inhibitor, or of cycloheximide, the translation inhibitor, on COX-2 modification. Specifically, the effect of low concentration of GS-HCl (1 mM) or of tunicamycin (0.1 g/ml) to produce the aglycosylated COX-2 was rescued by the proteasomal inhibitor MG132 but not by the lysosomal or caspase inhibitors. However, the proteasomal inhibitors did not show an effect at 5mM GS-HCl, which produced the aglycosylated or completely deglycosylated form of COX-2. Notably, GS-HCl (5 mM) also facilitated degradation of the higher molecular species of COX-2 in IL-1 -treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1 - or tumor necrosis factor- -driven COX-2 in differenthumancell lines, including Hep2 (bronchial) and H292 (laryngeal). However, GS-HCl did not affect COX-1 protein expression. These results demonstrate for the first time that GSHCl inhibits COX-2 activity by preventing COX-2 co-translational N-glycosylation and by facilitating COX-2 protein turnover during translation in a proteasome-dependent manner. Cyclooxygenase (COX),3 also referred prostaglandin (PG) H synthase, is the rate-limiting enzyme in the biosynthesis of PGs and related eicosanoids from arachidonic acid metabolism (1). Physiologically, PGs are involved in inflammatory response, bone development, wound healing, and the reproductive system. If excessive, however, PGs play a pathogenic role in many chronic inflammatory and neoplastic diseases (1, 2). In eukaryote cells, COX has two isoforms (1–3). COX-1 is constitutively expressed in most cells, and COX-1-derived PGs are involved in the maintenance of physiological functions. On the other hand, COX-2 is inducible by pro-inflammatory cytokines, tumor promoters, mitogenes, oncogenes, and growth factors in many types of cells, including monocytes, fibroblasts, and endothelial cells (1–5). Evidence that nonsteroidal anti-inflammatory drugs or compounds that target COX-2 lessen major inflammatory symptoms such as fever and pain suggests a role for COX-2 in inflammation (6). COX-2 expression is regulated at transcription, post-transcription, and translation. COX-2 transcription is induced by various exogenous stimuli that regulate intracellular signaling pathways that in turn modulate the activity of transcription factors and hence stimulate the COX-2 promoter (7). The cyclic AMP-responsive element, nuclear factor-interleukin 6, and NF- B cis-acting elements were shown to be important for transcriptional COX-2 induction (8, 9). Stabilization and nuclear export of COX-2 mRNA at post-transcriptional levels are also necessary for maximal COX-2 induction (10 –12). In addition, activities of MAPKs, including ERKs, p38 MAPK, and JNKs, were reported to be important for COX-2 expression (13, 14). COX-2 is an N-glycoprotein with four glycosylation sites (15, 16). Of interest, it has been previously shown that inhibition of COX-2 N-glycosy-lation by site-directed mutagenesis or tunicamycin (TN), a protein N-glycosylation inhibitor, results in expression of COX-2 with the reduced molecular mass and activity (17), indicating the importance of this co-translational modification in COX-2 enzyme catalysis. Glucosamine (GS) is an amino monosaccharide and has been widely used as an alternative regimen for rheumatoid arthritis or osteoarthritis. Recent in vivo studies have shown that GS salts, including GS sulfate or GS-HCl, have preventive actions on adjuvant arthritis in rats (18), possess the significant symptom- modifying effect on osteoarthritis in long term human clinical trials (19), and reduce equine cartilage degradation (20). Moreover, many recent in vitro studies have demonstrated that GS-HCl suppresses IL-1 -induced COX-2 expression by decreasing COX-2 transcript level in chondrocytes and synoviocytes (21), and that GS sulfate inhibits IL-1 -induced NF- B activation in human osteoarthritic chondrocytes (22) and decreases TNF- - and interferon- -induced ICAM-1 (intercellular adhesion molecule 1) expression at transcriptional level in human retinal pigment epithelial cells (23). From these, it is suggested that GS exerts its anti-inflammatory effect in part through transcriptional down-regulation of various genes involved in inflammation, cell adhesion, matrix degradation, and/or migration. However, the action mechanism by which GS affects expression and activity of COX-2 is not fully understood. In this study, we evaluated the effects of different GS salts (GS-HCl, GS sulfate) or a GS derivative (N-acetyl GS) and galactosamine HCl (Gal-HCl), another hexosamine, on the expression of COX-2 and production of PGE2 by IL-1 in A549 human lung epithelial cells. Here we demonstrate for the first time a new mechanism of GS-HCl to specifically inhibit endogenous and agonist-driven COX-2 at protein level.