CYSTEINE PROTEINASES IN GH(4)C(1) CELLS, A RAT PITUITARY-TUMOR CELL-LINE, ARE SECRETED BY THE CONSTITUTIVE AND REGULATED SECRETORY PATHWAYS

Secretory granules of GH(4)C(1) cells, a rat pituitary tumor cell line , are known to be induced by the treatment of estradiol (E(2)), insuli n, and epidermal growth factor (EGF). We examined changes in the local ization of cathepsins B, H, and L, lysosomal cysteine proteinases, in GH(4)C(1) cells before and after hormonal treatment. Northern blotting and immunofluorescence microscopy showed that both mRNAs and intracel lular protein concentrations of these enzymes were increased in the ho rmone-induced cells, By immunoelectron microscopy, immunogold particle s indicating cathepsins B, H, and L were localized not only in lysosom es but also in some secretory granules. To further examine the molecul ar forms of these proteinases in secretory granules, radiolabeling and immunoprecipitation methods were applied to the media of the cells in cubated with or without secretagogues (100 nM 12-O-tetradecanoylphorbo l-13-acetate and 50 mu M forskolin); the proforms of cathepsins B, H, and L were secreted from the cells by the constitutive pathway, wherea s the mature forms of cathepsins B and H, and the preform and mature f orm of cathepsin L were secreted by the regulated pathway. These resul ts suggest that in hormone-induced GH(4)C(1) cells, cathepsins B, H, a nd L are sorted from the Golgi complex not only into lysosomes but als o into secretory granules, in which proforms of cathepsins B and H, an d a part of procathepsin L are processed into mature forms.