Collagenase synthesis by cloned rabbit pulp cells--molecular and immunological identity of pulp cell and fibroblast enzyme.
Cultures of cloned rabbit pulp (RP) cells without stimulation produced collagenase of a concentration as high as reference rabbit skin fibroblast cultures which were stimulated with phorbol myristate acetate (PMA, 100 ng/ml). The RP cell collagenase was compared with reference fibroblast collagenase in Western blot analysis using monoclonal antibodies prepared against RP cell collagenase and a polyclonal antibody prepared against rabbit fibroblast collagenase. Both enzyme preparations revealed, with either antibody, identical bands of approximate molecular masses 57,000, 52,500, and 45,000. These antibody preparations variously inhibited RP cell collagenase activity. Intracellular collagenase in RP cells in culture was demonstrated by the indirect immunofluorescence antibody technique using polyclonal anti-fibroblast collagenase antibody. RNA samples from RP cells hybridized with rabbit fibroblast collagenase cDNA (clone H9) and showed a distinct band at 2.7 kb. Both control and PMA-stimulated RP cells and PMA-stimulated reference skin fibroblasts demonstrated strong cytoplasmic hybridization between H9 and collagenase mRNA. The results indicate that RP cell collagenase is identical to rabbit fibroblast collagenase, and that the RP cell line provides a useful in vitro reference system for the study of collagenolysis in the rabbit model.
Goldberg
L
L
Laboratory for the Study of Connective Tissue Metabolism, Harvard School of Dental Medicine, Boston, Massachusetts 02115.
Sakamoto
M
M
Bolender
Y
Y
Sakamoto
S
S
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D002999:Clone Cells; D015342:DNA Probes; D003782:Dental Pulp / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D003012:Microbial Collagenase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011061:Poly A / Q000302:isolation & purification; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits
D015342:DNA Probes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D003012:Microbial Collagenase