Cysteine proteinases in GH4C1 cells, a rat pituitary tumor cell line, are secreted by the constitutive and regulated secretory pathways.
Secretory granules of GH4C1 cells, a rat pituitary tumor cell line, are known to be induced by the treatment of estradiol (E2), insulin, and epidermal growth factor (EGF). We examined changes in the localization of cathepsins B, H, and L, lysosomal cysteine proteinases, in GH4C1 cells before and after hormonal treatment. Northern blotting and immunofluorescence microscopy showed that both mRNAs and intracellular protein concentrations of these enzymes were increased in the hormone-induced cells. By immunoelectron microscopy, immunogold particles indicating cathepsins B, H, and L were localized not only in lysosomes but also in some secretory granules. To further examine the molecular forms of these proteinases in secretory granules, radiolabeling and immunoprecipitation methods were applied to the media of the cells incubated with or without secretagogues (100 nM 12-O-tetradecanoylphorbol-13-acetate and 50 microM forskolin); the proforms of cathepsins B, H, and L were secreted from the cells by the constitutive pathway, whereas the mature forms of cathepsins B and H, and the proform and mature form of cathepsin L were secreted by the regulated pathway. These results suggest that in hormone-induced GH4C1 cells, cathepsins B, H, and L are sorted from the Golgi complex not only into lysosomes but also into secretory granules, in which proforms of cathepsins B and H, and a part of procathepsin L are processed into mature forms.
Department of Cell Biology and Neuroanatomy, School of Medicine, Iwate Medical University, Morioka, Japan.
S
S
Waguri
N
N
Sato
T
T
Watanabe
K
K
Ishidoh
E
E
Kominami
K
K
Sato
Y
Y
Uchiyama
D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000378:metabolism; D056655:Cathepsin H; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D010450:Endopeptidases; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D008247:Lysosomes; D008297:Male; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007328:Insulin; D017527:Receptor, IGF Type 2; D004958:Estradiol; D004815:Epidermal Growth Factor; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534280:Ctsl protein, rat; D056655:Cathepsin H; C534128:Ctsh protein, rat