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
\ninvolved in many inflammatory processes. Glucosamine (GS)
\nis an amino monosaccharide and has been widely used for
\nalternative regimen of (osteo)arthritis. However, the mechanism
\nof action of GS on COX-2 expression remains unclear.
\nHere we describe a new action mechanism of glucosamine
\nhydrochloride (GS-HCl) to tackle endogenous and agonistdriven
\nCOX-2 at protein level. GS-HCl (but not GS sulfate,
\nN-acetyl GS, or galactosamine HCl) resulted in a shift in the
\nmolecular mass of COX-2 from 72–74 to 66–70 kDa and concomitant
\ninhibition of prostaglandin E2 production in a concentration-
\ndependent manner in interleukin (IL)-1 -treated
\nA549 human lung epithelial cells. Remarkably, GS-HCl-mediated
\ndecrease in COX-2 molecular mass was associated
\nwith inhibition of COX-2 N-glycosylation during translation,
\nas assessed by the effect of tunicamycin, the protein N-glycosylation
\ninhibitor, or of cycloheximide, the translation inhibitor,
\non COX-2 modification. Specifically, the effect of low
\nconcentration of GS-HCl (1 mM) or of tunicamycin (0.1
\n g/ml) to produce the aglycosylated COX-2 was rescued by
\nthe proteasomal inhibitor MG132 but not by the lysosomal or
\ncaspase inhibitors. However, the proteasomal inhibitors did not
\nshow an effect at 5mM GS-HCl, which produced the aglycosylated
\nor completely deglycosylated form of COX-2. Notably, GS-HCl (5
\nmM) also facilitated degradation of the higher molecular species of
\nCOX-2 in IL-1 -treated A549 cells that was retarded by MG132.
\nGS-HCl (5 mM) was also able to decrease the molecular mass of
\nendogenous and IL-1 - or tumor necrosis factor- -driven COX-2
\nin differenthumancell lines, including Hep2 (bronchial) and H292
\n(laryngeal). However, GS-HCl did not affect COX-1 protein
\nexpression. These results demonstrate for the first time that GSHCl
\ninhibits COX-2 activity by preventing COX-2 co-translational
\nN-glycosylation and by facilitating COX-2 protein turnover during
\ntranslation in a proteasome-dependent manner.
\nCyclooxygenase (COX),3 also referred prostaglandin (PG)
\nH synthase, is the rate-limiting enzyme in the biosynthesis of
\nPGs and related eicosanoids from arachidonic acid metabolism
\n(1). Physiologically, PGs are involved in inflammatory
\nresponse, bone development, wound healing, and the reproductive
\nsystem. If excessive, however, PGs play a pathogenic
\nrole in many chronic inflammatory and neoplastic diseases
\n(1, 2).
\nIn eukaryote cells, COX has two isoforms (1–3). COX-1 is
\nconstitutively expressed in most cells, and COX-1-derived
\nPGs are involved in the maintenance of physiological functions.
\nOn the other hand, COX-2 is inducible by pro-inflammatory
\ncytokines, tumor promoters, mitogenes, oncogenes,
\nand growth factors in many types of cells, including monocytes,
\nfibroblasts, and endothelial cells (1–5). Evidence that
\nnonsteroidal anti-inflammatory drugs or compounds that
\ntarget COX-2 lessen major inflammatory symptoms such as
\nfever and pain suggests a role for COX-2 in inflammation (6).
\nCOX-2 expression is regulated at transcription, post-transcription,
\nand translation. COX-2 transcription is induced
\nby various exogenous stimuli that regulate intracellular signaling
\npathways that in turn modulate the activity of transcription
\nfactors and hence stimulate the COX-2 promoter
\n(7). The cyclic AMP-responsive element, nuclear factor-interleukin
\n6, and NF- B cis-acting elements were shown to be
\nimportant for transcriptional COX-2 induction (8, 9). Stabilization
\nand nuclear export of COX-2 mRNA at post-transcriptional
\nlevels are also necessary for maximal COX-2
\ninduction (10 –12). In addition, activities of MAPKs, including
\nERKs, p38 MAPK, and JNKs, were reported to be important
\nfor COX-2 expression (13, 14). COX-2 is an N-glycoprotein
\nwith four glycosylation sites (15, 16). Of interest, it has
\nbeen previously shown that inhibition of COX-2 N-glycosy-lation by site-directed mutagenesis or tunicamycin (TN), a
\nprotein N-glycosylation inhibitor, results in expression of
\nCOX-2 with the reduced molecular mass and activity (17),
\nindicating the importance of this co-translational modification
\nin COX-2 enzyme catalysis.
\nGlucosamine (GS) is an amino monosaccharide and has been
\nwidely used as an alternative regimen for rheumatoid arthritis
\nor osteoarthritis. Recent in vivo studies have shown that GS
\nsalts, including GS sulfate or GS-HCl, have preventive actions
\non adjuvant arthritis in rats (18), possess the significant symptom-
\nmodifying effect on osteoarthritis in long term human
\nclinical trials (19), and reduce equine cartilage degradation (20).
\nMoreover, many recent in vitro studies have demonstrated that
\nGS-HCl suppresses IL-1 -induced COX-2 expression by
\ndecreasing COX-2 transcript level in chondrocytes and synoviocytes
\n(21), and that GS sulfate inhibits IL-1 -induced NF- B
\nactivation in human osteoarthritic chondrocytes (22) and
\ndecreases TNF- - and interferon- -induced ICAM-1 (intercellular
\nadhesion molecule 1) expression at transcriptional level in
\nhuman retinal pigment epithelial cells (23). From these, it is
\nsuggested that GS exerts its anti-inflammatory effect in part
\nthrough transcriptional down-regulation of various genes
\ninvolved in inflammation, cell adhesion, matrix degradation,
\nand/or migration. However, the action mechanism by which
\nGS affects expression and activity of COX-2 is not fully
\nunderstood.
\nIn this study, we evaluated the effects of different GS salts
\n(GS-HCl, GS sulfate) or a GS derivative (N-acetyl GS) and
\ngalactosamine HCl (Gal-HCl), another hexosamine, on the
\nexpression of COX-2 and production of PGE2 by IL-1 in A549
\nhuman lung epithelial cells. Here we demonstrate for the first
\ntime a new mechanism of GS-HCl to specifically inhibit endogenous
\nand agonist-driven COX-2 at protein level.