Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture.
The effect of hyaluronidase treatment on the incorporation of [3H]glucosamine into hyaluronate in human skin fibroblast cultures was investigated. Fourth passage cells in confluent cultures were treated with hyaluronidase from bovine tests, Streptomyces and leech in Dulbecco's minimum essential medium in the presence of 3% fetal calf serum. The medium was removed from the control (non-treated) and the treated cultures and the washed cell layers were incubated with [3H]glucosamine and [35S]sulfate. [3H]Hyaluronate was separated by DEAE Trisacyl chromatography and identified by specific enzymic assays. Hyaluronidase treatment induced an increase in the amount of labelled hyaluronate secreted into the medium and into the pericellular compartment. This amount reached a plateau with increasing enzyme concentration and with the time of treatment. Oligosaccharides derived from hyaluronate did not produce this effect. The maximal increase was about 3-fold, and was not inhibited by exogenous hyaluronate (25-100 micrograms/ml) or by oligosaccharides from hyaluronate. Cycloheximide (0.03 mM) inhibited hyaluronate synthesis by 18% or less in the control cells and by 50% in the hyaluronidase-pretreated fibroblasts. No significant difference was found in the hyaluronate synthase activity between control and treated cells, at 60 min following treatment, indicating the reversibility of the effect. The persistence of the stimulation required the presence of hyaluronidase. The treatment of cells with specific hyaluronidases (from Streptomyces and leech) or with testicular hyaluronidase did not modify the labelling of the sulfated glycosaminoglycans. The incorporation kinetics of the [3H]glucosamine into labeled hyaluronate and the increased amount of non-labelled hyaluronate determined by radiometric assay indicated a specific stimulation of hyaluronate synthesis in the hyaluronidase-pretreated fibroblast cultures.
Larnier
C
C
Laboratoire de Biochimie du Tissu Conjonctif, UA CNRS 1174, Faculté de Médecine, Université Paris XII, Creteil, France.
Kerneur
C
C
Robert
L
L
Moczar
M
M
D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D016695:Glycosyltransferases; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology*; D007700:Kinetics; D007865:Leeches / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins; D013302:Streptomyces / Q000201:enzymology; D013431:Sulfates / Q000378:metabolism; D013737:Testis / Q000201:enzymology*; D014166:Transferases; D029867:Xenopus Proteins
D003470:Culture Media; D008565:Membrane Proteins; D013431:Sulfates; D029867:Xenopus Proteins; D006820:Hyaluronic Acid; D003513:Cycloheximide; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases; D006821:Hyaluronoglucosaminidase; D005944:Glucosamine