Alexander R. Shikhman

N-Acetylglucosamine Prevents IL-1β-Mediated Activation of Human Chondrocytes

Glucosamine represents one of the most commonly used drugs to treat osteoarthritis. However, mechanisms of its antiarthritic activities are still poorly understood. The present study identifies a novel mechanism of glucosamine-mediated anti-inflammatory activity. It is shown that both glucosamine and N-acetylglucosamine inhibit IL-1β- and TNF-α-induced NO production in normal human articular chondrocytes. The effect of the sugars on NO production is specific, since several other monosaccharides, including glucose, glucuronic acid, and N-acetylmannosamine, do not express this activity. Furthermore, N-acetylglucosamine polymers, including the dimer and the trimer, also do not affect NO production. The observed suppression of IL-1β-induced NO production is associated with inhibition of inducible NO synthase mRNA and protein expression. In addition, N-acetylglucosamine also suppresses the production of IL-1β-induced cyclooxygenase-2 and IL-6. The constitutively expressed cyclooxygenase-1, however, was not affected by the sugar. N-acetylglucosamine-mediated inhibition of the IL-1β response of human chondrocytes was not associated with the decreased inhibition of the mitogen-activated protein kinases c-Jun N-terminal kinase, extracellular signal-related kinase, and p38, nor with activation of the transcription factor NF-κB. In conclusion, these results demonstrate that N-acetylglucosamine expresses a unique range of activities and identifies a novel mechanism for the inhibition of inflammatory processes.

Cytokine Regulation of Facilitated Glucose Transport in Human Articular Chondrocytes

Glucose serves as the major energy substrate and the main precursor for the synthesis of glycosaminoglycans in chondrocytes. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. This study examines molecular regulation of facilitated glucose transport in normal human articular chondrocytes by proinflammatory cytokines. IL-1β and TNF-α, and to a lesser degree IL-6, accelerate facilitated glucose transport as measured by [3H]2-deoxyglucose uptake. IL-1β induces an increased expression of glucose transporter (GLUT) 1 mRNA and protein, and GLUT9 mRNA. GLUT3 and GLUT8 mRNA are constitutively expressed in chondrocytes and are not regulated by IL-1β. GLUT2 and GLUT4 mRNA are not detected in chondrocytes. IL-1β stimulates GLUT1 protein glycosylation and plasma membrane incorporation. IL-1β regulation of glucose transport in chondrocytes depends on protein kinase C and p38 signal transduction pathways, and does not require phosphoinositide 3-kinase, extracellular signal-related kinase, or c-Jun N-terminal kinase activation. IL-1β-accelerated glucose transport in chondrocytes is not mediated by endogenous NO or eicosanoids. These results demonstrate that stimulation of glucose transport represents a component of the chondrocyte response to IL-1β. Two classes of GLUTs are identified in chondrocytes, constitutively expressed GLUT3 and GLUT8, and the inducible GLUT1 and GLUT9.

Role of nitric oxide, reactive oxygen species, and p38 MAP kinase in the regulation of human chondrocyte apoptosis

This study addresses mechanisms by which interleukin‐1β (IL‐1β) regulates human chondrocyte apoptosis induced by a combination of the anti‐CD95 antibody CH‐11 and the proteasome inhibitor (PSI). The effect of IL‐1β on apoptosis varied among tissue samples. IL‐1β either enhanced (16/22 samples) or inhibited (6/22 samples) DNA fragmentation and caspase‐3 processing. The protective effect of IL‐1β was abrogated by the nitric oxide (NO) synthesis inhibitor N‐monomethyl‐l‐arginine (L‐NMMA) while apoptosis stimulation was not affected. The NO‐donors sodium nitroprusside (SNP) and S‐nitroso‐N‐acetyl penicillamine (SNAP) blocked DNA fragmentation, and this was associated with partial inhibition of caspase‐3 processing. Pyrrolidine dithiocarbamate (PDTC), a scavenger of reactive oxygen species (ROS) blocked apoptosis induction by CH‐11/PSI as well as the enhancement by IL‐1β. The pro‐apoptotic effects of IL‐1β were also abrogated by the p38 inhibitor SB 202190. In conclusion, IL‐1β augments CH‐11/PSI induced apoptosis in the majority of chondrocyte samples. The pro‐apoptotic effect of IL‐1β is not dependent on NO. In contrast, the anti‐apoptotic effect of IL‐1β observed in a minority of samples is partially NO‐dependent. J. Cell. Physiol. 197: 379–387, 2003© 2003 Wiley‐Liss, Inc.

Profile of glycosaminoglycan-degrading glycosidases and glycoside sulfatases secreted by human articular chondrocytes in homeostasis and inflammation.

OBJECTIVE To determine enzymatic activities of the 8 key glycosaminoglycan-degrading glycosidases and glycoside sulfatases in cultured human articular chondrocytes and in synovial fluid from patients with osteoarthritis. METHODS The following enzymes were analyzed: hexosaminidase and its isoenzyme A, N-acetyl-alpha-D-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-L-iduronidase, aryl sulfatase, and galactose-6-sulfate sulfatase. Activity of the selected enzymes was analyzed by fluorometry with the aid of 4-methylumbelliferryl derivatives of the appropriate monosaccharides. RESULTS Hexosaminidase was found to be the dominant enzyme released by chondrocytes into the extracellular compartment. Stimulation of chondrocytes with interleukin-1beta resulted in a selective increase of the extracellular hexosaminidase activity and, to a lesser degree, of the extracellular beta-galactosidase activity, without significant changes in the activity of the other studied enzymes. Analysis of the pH dependency of the enzymatic activities revealed that even at neutral pH, hexosaminidase expressed a measurable activity, much higher than the activity of the other studied enzymes. Chondrocyte apoptosis did not result in increased extracellular glycosidase activities, including hexosaminidase activity. The spectrum of glycosidase and glycoside sulfatase activities in the synovial fluid from patients with osteoarthritis was similar to that in cultured human articular chondrocytes. CONCLUSION These data support the concept that lysosomal glycosidases, in particular hexosaminidase, represent a distinct subset of cartilage matrix-degrading enzymes that are activated by proinflammatory stimuli.

Hexosaminidase inhibitors as new drug candidates for the therapy of osteoarthritis

BACKGROUND Articular cartilage from patients with osteoarthritis is characterized by a decreased concentration and reduced size of glycosaminoglycans. Degeneration of the cartilage matrix is a multifactorial process, which is due in part to accelerated glycosaminoglycan catabolism. Recently, we have demonstrated that hexosaminidase represents the dominant glycosaminoglycan-degrading glycosidase released by chondrocytes into the extracellular compartment and is the dominant glycosidase in synovial fluid from patients with osteoarthritis. Inhibition of hexosaminidase activity may represent a novel approach to the prevention of cartilage matrix glycosaminoglycan degradation and a potentially new strategy to treat osteoarthritis. RESULTS We have synthesized and investigated a series of iminocyclitols designed as transition-state analog inhibitors of human hexosaminidase, and demonstrated that the five-membered iminocyclitol 4 expresses the strongest inhibitory activity with K(i)=24 nM. Inhibition of hexosaminidase activity in human cultured articular chondrocytes and human chondrosarcoma cells with iminocyclitol 4 resulted in accumulation of hyaluronic acid and sulfated glycosaminoglycans in the cell-associated fraction. Similarly, incubation of human cartilage tissue with iminocyclitol 4 resulted in an accumulation of glycosaminoglycans in the pericellular compartment. CONCLUSIONS Inhibition of hexosaminidase activity represents a new strategy for preventing or even reversing cartilage degradation in patients with osteoarthritis.

Differential metabolic effects of glucosamine and N-acetylglucosamine in human articular chondrocytes

OBJECTIVE Aminosugars are commonly used to treat osteoarthritis; however, molecular mechanisms mediating their anti-arthritic activities are still poorly understood. This study analyzes facilitated transport and metabolic effects of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) in human articular chondrocytes. METHODS Human articular chondrocytes were isolated from knee cartilage. Facilitated transport of glucose, GlcN and GlcNAc was measured by uptake of [3H]2-deoxyglucose, [3H]GlcN and [3H]GlcNAc. Glucose transporter (GLUT) expression was analyzed by Western blotting. Production of sulfated glycosaminoglycans (SGAG) was measured using [(35)S]SO4. Hyaluronan was quantified using hyaluronan binding protein. RESULTS Chondrocytes actively import and metabolize GlcN but not GlcNAc and this represents a cell-type specific phenomenon. Similar to facilitated glucose transport, GlcN transport in chondrocytes is accelerated by cytokines and growth factors. GlcN non-competitively inhibits basal glucose transport, which in part depends on GlcN-mediated depletion of ATP stores. In IL-1beta-stimulated chondrocytes, GlcN inhibits membrane translocation of GLUT1 and 6, but does not affect the expression of GLUT3. In contrast to GlcN, GlcNAc accelerates facilitated glucose transport. In parallel with the opposing actions of these aminosugars on glucose transport, GlcN inhibits hyaluronan and SGAG synthesis while GlcNAc stimulates hyaluronan synthesis. GlcNAc-accelerated hyaluronan synthesis is associated with upregulation of hyaluronan synthase-2. CONCLUSION Differences in GlcN and GlcNAc uptake, and their subsequent effects on glucose transport, GLUT expression and SGAG and hyaluronan synthesis, indicate that these two aminosugars have distinct molecular mechanisms mediating their differential biological activities in chondrocytes.