EXPRESSION OF EXTRACELLULAR-MATRIX DEGRADING METALLOPROTEINASES BY CULTURED HUMAN CYTOTROPHOBLAST CELLS - EFFECTS OF CELL-ADHESION AND IMMUNOPURIFICATION

In vitro, invasion of basement membrane by human trophoblast can be blocked by metalloproteinase inhibitors. The purpose of our study was to characterize these enzymes by zymography, to define their cellular origin. First-trimester cytotrophoblast cells were prepared according to the method of Kliman et al. Half of the cell suspension was further purified with an antibody to leukocyte common antigen (CD45). Cytotrophoblast cells (immunopurified or not) were incubated in Dulbecco's modified Eagle's medium on different matrices. Progesterone, total human chorionic gonadotropin, and free beta-human chorionic gonadotropin were measured in the supernatant by radioimmunoassay or enzyme immunoassays. Secreted (in the medium) and cell-bound proteases were characterized by zymography on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Cytotrophoblast cell preparations contained 12% to 34% leukocyte common antigen-positive cells before and 0% after immunopurification. Large zones of digested matrices were observed after 48 hours of culture on Matrigel or rat tail collagen but not on agarose. Cells secreted progesterone, human chorionic gonadotropin, and free beta-human chorionic gonadotropin in vitro, but no difference was observed among cells grown on different matrices or between immunopurified and nonimmunopurified cells. By zymography, seven gelatin-degrading enzymes were seen in culture supernatants and five of them were present in cell lysates. The molecular weights of these proteases ranged from 59 to 230 kd. Immunopurification eliminated three of these enzymes, so they were clearly produced by bone marrow-derived cells (leukocyte common antigen positive) contaminating the cytotrophoblast cell preparation. Cells grown on Matrigel express a unique 59 kd gelatinase that was not seen in the supernatants of cells grown on other matrices. Zymography in the presence of inhibitors showed that these enzymes were neutral metalloproteinases, which might be responsible for the observed extracellular matrix degradation.