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

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Author(s)
Byeong-Churl JangSu-Haeng SungJong-Gu ParkJong-Wook ParkJae Hoon BaeDong Hoon ShinGi-Young ParkSeung-Bum HanSeong-Il Suh
Keimyung Author(s)
Park, Gi YoungHan, Sung BeomJang, Byeong ChurlSuh, Seong IlShin, Dong HoonBae, Jae HoonPark, Jong WookPark, Jong Gu
Department
Dept. of Rehabilitation Medicine (재활의학)
Dept. of Internal Medicine (내과학)
Dept. of Molecular Medicine (분자의학)
Dept. of Microbiology (미생물학)
Dept. of Preventive Medicine (예방의학)
Dept. of Physiology (생리학)
Dept. of Immunology (면역학)
Institute for Medical Science (의과학연구소)
Journal Title
Journal of Biological Chemistry
Issued Date
2007
Volume
282
Issue
38
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.
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