tretinoin and mannosamine

To investigate the effect of long-term exposure to glucosamine or mannosamine on the catabolism of aggrecan by explant cultures of bovine articular cartilage maintained in the presence of retinoic acid.. The kinetics of loss of 35S-labeled and total aggrecan from explant cultures of bovine articular cartilage maintained in the presence of 1 micro M retinoic acid and exposed to varying concentrations of glucosamine or mannosamine was investigated over a 9-day culture period. In other experiments, the reversibility of the inhibition of aggrecan catabolism by glucosamine or mannosamine was investigated in cultures exposed to these amino sugars for the first 5 days of a 15-day culture period. The metabolism of chondrocytes exposed to these amino sugars was evaluated by measurement of lactate production or 3H-serine and 35S-sulfate incorporation into protein and glycosaminoglycans, respectively. The direct effect of these amino sugars on soluble aggrecanase activity was determined from immunoblots of aggrecan digests.. Glucosamine at 5mM concentration and mannosamine at 2mM concentration inhibited degradation of radiolabeled and chemical levels of aggrecan. At concentrations of up to 10mM amino sugars, the metabolism of chondrocytes was not impaired, as determined by lactate production, protein synthesis and the incorporation of 35S-sulfate into proteoglycans. These amino sugars did not inhibit soluble aggrecanase activity. The exposure of articular cartilage explants to 5mM glucosamine or mannosamine for 5 days in culture in the presence or absence of retinoic acid did not provide long-term suppression of stimulated aggrecan loss.. This study indicates that continuous presence of amino sugars is required to protect cartilage from stimulated loss of aggrecan.

Topics: Aggrecans; Animals; Blotting, Western; Cartilage, Articular; Cattle; Culture Techniques; Endopeptidases; Extracellular Matrix Proteins; Glucosamine; Glycosaminoglycans; Hexosamines; Lactates; Lectins, C-Type; Protein Biosynthesis; Protein Denaturation; Proteoglycans; Solubility; Tretinoin

The control of chondrocyte-mediated degradation of aggrecan has been studied in rat chondrosarcoma cells and bovine cartilage explants treated with either IL-1 or retinoic acid. The capacity of glucosamine to inhibit the aggrecanase-mediated response (J. D. Sandy, D. Gamett, V. Thompson, and C. Verscharen (1998) Biochem. J. 335, 59-66) has been extended to an investigation of the effect of other hexosamines. Mannosamine inhibits the aggrecanase response to both IL-1 and RA at about one-tenth the concentration of glucosamine in both rat cell and bovine explant systems. This effect of mannosamine appears to be due to its capacity to inhibit the synthesis of glycosylphosphatidylinositol (GPI)-linked proteins by chondrocytes since the GPI synthesis inhibitor 2-deoxyfluoroglucose (2-DFG) also inhibited the aggrecanase response to IL-1b and RA in rat cells. Moreover, phosphatidylinositol-specific phospholipase C (PIPLC) treatment of rat cells markedly inhibited the aggrecanase response to IL-1b and RA. These inhibitory effects of mannosamine, 2-DFG, and PIPLC in rat cells did not appear to be due to an interference with general biosynthetic activity of the cells as measured by [3H]proline incorporation into secreted proteins. We suggest that the aggrecanase response by chondrocytes to IL-1 and RA is dependent on the activity of a GPI-anchored protein on the chondrocyte cell surface.

Topics: Aggrecans; Animals; Cartilage; Cattle; Chondrocytes; Culture Techniques; Deoxyglucose; Dose-Response Relationship, Drug; Endopeptidases; Extracellular Matrix Proteins; Glycosylphosphatidylinositols; Hexosamines; Interleukin-1; Lectins, C-Type; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Proteoglycans; Rats; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases