Abstract
COX-2 and its products, including prostaglandin E(2), 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 agonist-driven 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 E(2) production in a concentration-dependent manner in interleukin (IL)-1beta-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 microg/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 5 mM 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-1beta-treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1beta- or tumor necrosis factor-alpha-driven COX-2 in different human cell 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 GS-HCl 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.
Highlights
In eukaryote cells, COX has two isoforms [1,2,3]
We here report for the first time that among GS salts or derivative and other hexosamine tested, GSHCl inhibits endogenous and cytokine-driven COX-2 expression at protein level via a mechanism associated with the proteasome-dependent down-regulation of COX-2 N-glycosylation and turnover
It has been reported that glucosamine hydrochloride (GS-HCl) or GS sulfate inhibits IL-1-induced COX-2 expression by transcriptional down-regulation in chondrocytes, and the down-regulation is in part associated with the ability of GS-HCl to inhibit NF-B [22]
Summary
COX has two isoforms [1,2,3]. COX-1 is constitutively expressed in most cells, and COX-1-derived PGs are involved in the maintenance of physiological functions. TN treatment concentration-dependently inhibited COX-2 glycosylation, resulting in expression of the low molecular mass of COX-2 (mostly ϳ66 kDa) (lanes 6 –9, marked with star), which is considered the fully agylcosylated COX-2, in IL-1-treated A549 cells.
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