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 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 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 E 2 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 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-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 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 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.

Prednisone inhibits the IL-1β-induced expression of COX-2 in HEI-OC1 murine auditory cells through the inhibition of ERK-1/2, JNK-1 and AP-1 activity

“…We, as well as others have previously reported that COX-2 expression is regulated at the post-transcriptional (mRNA stability) and/or translational (protein turnover) levels (35,(42)(43)(44). In the present study, however, experiments using Act D and CHX revealed that PDN did not affect the mRNA and protein stability of COX-2 in the IL-1β-treated HEI-OC1 cells (Fig.…”

“…AP-1 is another transcription factor that binds to AP-1 cis-acting element and stimulates COX-2 transcription (31), and its activation is largely dependent on the activity of upstream signaling effectors, such as ERK-1/2 and JNK-1/2 (32,33). It has been demonstrated that treatment with IL-1β induces the phosphorylation of MAPKs (ERK-1/2, JNK-1/2 and p38 MAPK) and the IκB-α proteolysisdependent activation of NF-κB, and their activation is critical for the cytokine-mediated induction of COX-2 expression in various types of cells (29)(30)(31)34,35). Of note, has also been demonstrated that treatment with IL-1β (1 ng/ml) for 15 min increases the phosphorylation of ERK-1/2 and JNK-1/2, but not that of p38 MAPK in HEI-OC1 cells (36).…”

“…3A and B, lanes 1-8). COX-2 expression is also controlled by the COX-2 mRNA turnover (35,42,43). When ongoing transcription was blocked by Act D, a transcriptional inhibitor, there was no difference in the COX-2 mRNA levels in the IL-1β-treated HEI-OC1 cells either in the absence or presence of PDN (Fig.…”

“…COX-2 expression is also regulated by protein turnover (35). This promptly led us to investigate whether PDN affects the stability of COX-2 protein in the IL-1β-treated HEI-OC1 cells.…”

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes

“…Glucosamine (GS) has been well established as a substitutable medical drug for rheumatoid arthritis or osteoarthritis (Reginster et al, 2001;Hua et al, 2005). Previous studies have demonstrated that GS regulates expression of COX-2 and production of PGE2 via IL-1β treatment in A549 human lung epithelial cells (Jang et al, 2007). Also, GS reduced IL-1β induced COX-2 expression at both transcriptional and translational levels in chondrocytes and synoviocytes (Nakamura et al, 2004).…”