Jean Valbracht

Selective activation of the mitogen-activated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes.

Previous studies suggested that tyrosine kinase activation is an important signal transduction event in the IL-1 response of chondrocytes. The present study identifies the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 as major tyrosine phosphorylated proteins in IL-1 stimulated chondrocytes. Kinase assays on immunoprecipitates with myelin basic protein as substrate showed that ERK-1 and ERK-2 activation was detectable within 5 min after IL-1 stimulation and decreased to baseline within 60 min. Analysis of other members of the MAP kinase family showed that chondrocytes also express c-Jun NH2 terminal kinase (JNK)-1, JNK-2, and p38 proteins. These kinases were time-dependently activated by IL-1. Among other chondrocyte activators tested, only TNF activated all three of the MAP kinase subgroups. JNK and p38 were not activated by any of the other cytokines and growth factors tested. However, ERK was also activated by PDGF, IGF-1, and IL-6. Phorbol 12-myristate 13-acetate, calcium ionophore, and cAMP analogues only increased ERK activity but had no significant effects on JNK or p38. These results suggest differential activation of MAP kinase subgroups by extracellular stimuli. ERK is activated in response to qualitatively diverse extracellular stimuli and various second messenger agonists. In contrast, JNK and p38 are only activated by IL-1 or TNF, suggesting that these kinases participate in the induction of the catabolic program in cartilage.

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.

The osteoprotegerin/receptor activator of nuclear factor κB/receptor activator of nuclear factor κB ligand system in cartilage

OBJECTIVE The receptor activator of nuclear factor kappaB (RANK) is a member of the tumor necrosis factor receptor family. It is activated by the secreted or cell surface-bound RANK ligand (RANKL). Osteoprotegerin (OPG) is a soluble nonsignaling receptor for RANKL and interferes with RANK activation. This receptor-ligand system regulates the differentiation of osteoclasts and dendritic cells. The present study examined human articular cartilage for the expression of these molecules and the role of RANKL in the regulation of chondrocyte function. METHODS Normal and osteoarthritic (OA) human articular cartilage was used for explant tissue culture or for isolation of chondrocytes and cell culture. Expression of RANK, RANKL, and OPG was analyzed by immunohistochemistry, Western blotting, or reverse transcription-polymerase chain reaction. Recombinant RANKL was added to cartilage or chondrocyte cultures, and gene expression, collagenase and nitric oxide production, and NF-kappaB activation were determined. RESULTS RANK, RANKL, and OPG messenger RNA (mRNA) were expressed in normal cartilage. By immunohistochemistry, RANK, RANKL, and OPG were detected in the superficial zone of normal cartilage. OA cartilage contained increased levels of OPG mRNA, and expression of the 3 proteins extended into the midzone of OA cartilage. OPG was detected by Western blotting, and was increased in response to interleukin-1beta stimulation. OPG, RANK, and RANKL protein were also detected in cultured chondrocytes. Addition of exogenous RANKL did not activate NF-kappaB, induce expression of genes encoding proinflammatory mediators in chondrocytes, or stimulate the production of collagenase and nitric oxide. CONCLUSION These results demonstrate the expression of OPG, RANK, and RANKL in cartilage. However, RANKL does not activate human articular chondrocytes.

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.

Gold sodium thiomalate and chloroquine inhibit cytokine production in monocytic THP-1 cells through distinct transcriptional and posttranslational mechanisms

Gold sodium thiomalate (GST), chloroquine (CQ), and metho- trexate have been widely used in the therapy of rheumatoid arthritis and other inflammatory conditions. Using the human monocytic cell line THP-1 we have analyzed effects of these drugs on cytokine production and intracellular signaling. GST and CQ were equally effective in reducing lipopolysaccharide (LPS)-induced IL-1β release while CQ was a more effective inhibitor of TNF-α production than GST. Methotrexate did not affect production of these cytokines. CQ reduced IL-1β mRNA expression and strongly inhibited phosphorylation of mitogen-activated protein kinase (MAPK) p38, and to a lesser extent c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. In contrast, GST did not affect cytokine mRNA expression or MAPK activation. However, GST selectively inhibited the activity of the interleukin-1 converting enzyme (ICE)/caspase-1. These data demonstrate that CQ inhibits IL-1β release from monocytes by interfering with pretranscriptional signaling and TNF-α release by posttranslational events whereas GST downregulates IL-1β secretion by interfering with posttranslational IL-1β processing.