Hidekazu Munakata

EFFECT OF 4-METHYLUMBELLIFERONE ON CELL-FREE SYNTHESIS OF HYALURONIC ACID

Human skin fibroblasts were cultured in the presence of 0.5 mM 4‐methylumbelliferone for 12 h, and cell‐free synthesis of hyaluronic acid was performed using membrane‐rich fraction from the cells. The preincubation of the cells with 4‐methylumbelliferone reduced hyaluronic acid synthesis to 15% of that of non‐preincubated cells, although its chain length was not changed. On the other hand, without preincubation of the cells with 4‐methylumbelliferone, hyaluronic acid synthesis was not changed even when 4‐methylumbelliferone was added directly to the reaction mixture. These results suggest that 4‐methylumbelliferone represses the expression of hyaluronic acid synthase on the cell surface.

CHARACTERIZATION OF BETA -D-XYLOSIDE-INITIATED GLYCOSAMINOGLYCAN SYNTHESIZED BY HUMAN SKIN FIBROBLASTS IN THE PRESENCE OF TUNICAMYCIN

Human skin fibroblasts were incubated with a fluorogenic xyloside, 4-methylumbelliferyl-β-D-xyloside (Xyl-MU), in the presence or absence of tunicamycin. The xyloside-initiated glycosaminoglycans (GAG-MUs) were isolated from the culture medium, and their structures characterized. When the cells were incubated with Xyl-MU in the presence of 0.2 μg ml−1 tunicamycin, the synthesis of GAG-MU was increased about three fold, compared with the control value in the absence of tunicamycin (cells exposed to Xyl-MU alone). The structures of GAG-MUs synthesized in the presence or absence of tunicamycin were compared by HPLC analysis using gel-filtration and ion-exchange columns, enzymatic digestion, and unsaturated disaccharide composition analysis. The data indicated that cells incubated with tunicamycin produced more undersulfated and shorter GAG-MUs than cells without tynicamycin. These results suggest that tunicamycin inhibits the elongation and sulfation of glycosaminoglycan (GAG) chains and that, as a result, GAG-MUs with shorter chains and undersulfated residues, but possessing a large number of GAG chains, are synthesized in the presence of tunicamycin.