Collagenase production by early and late passage cultures of human fibroblasts.
Comparison of the proteins secreted by early and late passage cell cultures of human fibroblasts revealed a high level of immunoreactive collagenase (Mr = 55,000 Da and 58,000 Da) in the late passage cell culture conditioned medium. Both molecular weight species reacted with a monoclonal anticollagenase antibody and were apparently glycosylation varaents of the same protein. The question of whether the apparent age-dependent differences in collagenase synthesis reflected changes in protein synthesis or secretion was addressed by assaying immunoreactive collagenase and collagenase mRNA. Immunofluorescence microscopy of cellular collagenase revealed that the percentage of collagenase positive cells ranged from 1 to 6% (early passage) to 35 to 46% (late passage) indicating that the late passage cells had higher basal levels of collagenase synthesis. Later passage cultures also secreted higher levels of immunoprecipitable collagenase into the culture medium and Northern analysis established that the basal level of collagenase mRNA was also 10 times greater in late passage cells. High basal levels of collagenase were also observed in fibroblasts cultured from an in vivo aged donor and from donors with Werner's syndrome. Collagenase production was induced in both early and late passage cell cultures by exposure to fibroblast extracellular matrix, fibroblast conditioned media, polypeptide growth factors, or phorbol esters. The induced levels were always greater in the late passage cell cultures than in the early passage cell cultures.
Millis
A J
AJ
Department of Biological Sciences, University at Albany, State University of New York 12222.
Sottile
J
J
Hoyle
M
M
Mann
D M
DM
Diemer
V
V
D015152:Blotting, Northern; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D003012:Microbial Collagenase / Q000096:biosynthesis*; D010703:Phorbol Esters / Q000494:pharmacology
D006133:Growth Substances; D010703:Phorbol Esters; D003012:Microbial Collagenase