Waguri S, Sato N, Watanabe T, Ishidoh K, Kominami E, Sato K, Uchiyama Y
Department of Cell Biology and Neuroanatomy, School of Medicine, Iwate Medical University, Morioka, Japan.
Eur J Cell Biol. 1995 Aug;67(4):308-18.
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.
已知大鼠垂体瘤细胞系GH4C1细胞的分泌颗粒可由雌二醇（E2）、胰岛素和表皮生长因子（EGF）处理诱导产生。我们检测了激素处理前后GH4C1细胞中组织蛋白酶B、H和L（溶酶体半胱氨酸蛋白酶）定位的变化。Northern印迹法和免疫荧光显微镜检查显示，这些酶的mRNA和细胞内蛋白浓度在激素诱导的细胞中均增加。通过免疫电子显微镜观察，指示组织蛋白酶B、H和L的免疫金颗粒不仅定位于溶酶体，还定位于一些分泌颗粒中。为了进一步检测分泌颗粒中这些蛋白酶的分子形式，将放射性标记和免疫沉淀方法应用于用或不用促分泌剂（100 nM 12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯和50 μM福斯可林）孵育的细胞培养基中；组织蛋白酶B、H和L的前体形式通过组成型途径从细胞中分泌出来，而组织蛋白酶B和H的成熟形式以及组织蛋白酶L的前体形式和成熟形式则通过调节型途径分泌。这些结果表明，在激素诱导的GH4C1细胞中，组织蛋白酶B、H和L不仅从高尔基体复合体分选到溶酶体中，还分选到分泌颗粒中，在分泌颗粒中组织蛋白酶B和H的前体形式以及部分组织蛋白酶L原被加工成成熟形式。
