Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The
conditioned medium (CM) of fetal human astrocytes and five
glioma cell lines did not degrade
azocoll in
suspension, but several proteolytic activities, inhibitable by
1,10-phenanthroline, were detected on
sodium dodecyl sulfate-
polyacrylamide gels containing
gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the
glioma lines secreted an additional
proteinase (Mr 92,000).
After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000
proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000
proteinases bound specifically to a
gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000
proteinase was found to degrade
type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase
IgG.
Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]
methionine-labeled
proteins with anti-
tissue inhibitor of metalloproteinases IgG. The
glioma lines also secreted various amounts of two smaller inhibitors of
metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (
IMP-1 at Mr 22,000 and
IMP-2 at Mr 19,000), and an inhibitor not previously identified (
IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of
tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the
glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from
proteinases, the isolated
proteinases had activity against
azocoll and the
glycoprotein and
collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of
metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as
inflammation or local invasion of astrocytic
neoplasms.