PHORBOL ESTER-INDUCED CELL-SURFACE ASSOCIATION OF MATRIX METALLOPROTEINASE-9 IN HUMAN MCF1OA BREAST EPITHELIAL-CELLS

Cell surface association of extracellular matrix (ECM)-degrading enzym es has been suggested to facilitate proteolysis of ECM in areas of cel l-matrix contacts and to be crucial for the process of tumor cell inva sion. Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family of endopeptidases that has been shown to play a critical role in hydr olysis of ECM components and has been localized on the surface of tumo r 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 h uman breast epithelial MCF10A cells treated with 12-O-tetradecanoylpho rbol-13-acetate (TPA). Surface biotinylation and immunoprecipitation w ith 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, where as in the media, only the M-r 92,000 form was detected, mostly in comp lex 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 prodom ain, indicating that both represent latent enzymes. Pulse-chase analys is and endoglycosidase H digestion of surface-biotinylated MMP-9 forms demonstrated that the M-r 85,000 species was endoglycosidase H sensit ive, suggesting targeting of the precursor form of MMP-9 to the cell s urface. These studies demonstrate a specific cell surface association of MMP-9 in response to TPA that may help to localize TIMP-1-free enzy me on the surface of breast epithelial cells.