Detection and characterization, using fluoresceincadaverine, of amine acceptor protein substrates accessible to active transglutaminase expressed by rabbit articular chondrocytes.
The purpose of this study was to investigate the implication of transglutaminases in the biology of articular chondrocytes. Transglutaminase activity measurements performed on cell lysates showed that a transglutaminase was present in chondrocytes in primary culture and that it was strongly activated by limited proteolysis. In chondrocytes dedifferentiated by subculture or retinoic acid treatment, this transglutaminase appeared to be downregulated, while type II transglutaminase expression was induced. However, protein levels, mRNA steady-state levels or transglutaminase activity in whole-cell lysates do not necessarily reflect the activity present in living cells, as it is strongly regulated. Therefore, Fluoresceincadaverine, a fluorescent polyamine, was used for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells. After incubation of chondrocytes with Fluoresceincadaverine, dedifferentiated cells exhibited an extracellular labelling, while chondrocytes in primary culture did not, unless thrombin was added to the culture medium. In contrast, Fluoresceincadaverine labelling was not detected in the cytosol, although the transglutaminases were also partly cytosolic. By confocal microscopy and Western blot analysis of labelled cells in culture, fibronectin was shown to be the main substrate for both transglutaminases. The transglutaminases present in articular chondrocytes may, therefore, contribute to the organization and the stabilization of their extracellular matrix.
Lajemi
M
M
Laboratoire de Pharmacologie Cellulaire de l'Ecole Pratique des Hautes Etudes, Centre de Recherches Biomédicales des Cordeliers, Paris, France.
Demignot
S
S
Adolphe
M
M
D000588:Amines / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism*; D002358:Cartilage, Articular / Q000201:enzymology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005452:Fluoresceins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000096:biosynthesis / Q000378:metabolism*; D019204:GTP-Binding Proteins; D007527:Isoenzymes / Q000378:metabolism; D018613:Microscopy, Confocal; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013917:Thrombin / Q000494:pharmacology; D011503:Transglutaminases / Q000096:biosynthesis / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
D000588:Amines; D005452:Fluoresceins; D007527:Isoenzymes; D011506:Proteins; D014212:Tretinoin; D004492:Edetic Acid; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D013917:Thrombin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D002103:Cadaverine