Phorbol ester-induced cell surface association of matrix metalloproteinase-9 in human MCF10A breast epithelial cells.
Cell surface association of extracellular matrix (ECM)-degrading enzymes has been suggested to facilitate proteolysis of ECM in areas of cell-matrix contacts and to be crucial for the process of tumor cell invasion. Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family of endopeptidases that has been shown to play a critical role in hydrolysis of ECM components and has been localized on the surface of tumor cells. However, the nature of the cell surface association of MMP-9 is unknown. Here, we report the cell surface association of MMP-9 in human breast epithelial MCF10A cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Surface biotinylation and immunoprecipitation with anti-MMP-9 antibodies revealed the presence of two MMP-9 forms (M(r) 92,000 and 85,000) on the surface of TPA-treated MCF10A cells, whereas in the media, only the M(r) 92,000 form was detected, mostly in complex with TIMP-1, a specific MMP-9 inhibitor. The MMP-9 forms were also found in purified plasma membranes of TPA-treated cells. In contrast, the plasma membranes contained little or no TIMP-1. The surface-bound MMP-9 forms were recognized by an antibody to the NH2-terminal prodomain, indicating that both represent latent enzymes. Pulse-chase analysis and endoglycosidase H digestion of surface-biotinylated MMP-9 forms demonstrated that the M(r) 85,000 species was endoglycosidase H sensitive, suggesting targeting of the precursor form of MMP-9 to the cell surface. These studies demonstrate a specific cell surface association of MMP-9 in response to TPA that may help to localize TIMP-1-free enzyme on the surface of breast epithelial cells.
Toth
M
M
Department of Pathology and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
Gervasi
D C
DC
Fridman
R
R
D001940:Breast / Q000187:drug effects / Q000201:enzymology*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000737:chemistry; D004848:Epithelium / Q000201:enzymology; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D020780:Matrix Metalloproteinase 9; D008565:Membrane Proteins / Q000737:chemistry; D011480:Protease Inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019714:Tissue Inhibitor of Metalloproteinases
D017077:Culture Media, Conditioned; D006023:Glycoproteins; D008565:Membrane Proteins; D011480:Protease Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D006026:Glycoside Hydrolases; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; D013755:Tetradecanoylphorbol Acetate