Laurent-Matha V, Farnoud M R, Lucas A, Rougeot C, Garcia M, Rochefort H
INSERM Unité Hormones et Cancer (U 148), Université de Montpellier 1, 34090 Montpellier, France.
J Cell Sci. 1998 Sep;111 ( Pt 17):2539-49. doi: 10.1242/jcs.111.17.2539.
Cathepsin D trafficking is altered in cancer cells, leading to increased secretion of the pro-enzyme, which can be reinternalized by the same cancer cells and by stromal cells. We studied pro-cathepsin D endocytosis in two human breast cancer cell lines (MDA-MB231, MCF-7) and in human normal fibroblasts. Pro-enzyme uptake was studied indirectly through immunofluorescence analysis of anti-pro-cathepsin D monoclonal antibodies internalized in living cells. Both cancer cell lines internalized the pro-cathepsin D-antibody complex into endosomal compartments in the presence of 10 mM mannose-6-phosphate. Non-malignant fibroblasts, which do not secrete pro-cathepsin D, only internalized anti-cathepsin D antibody when purified pro-cathepsin D was added and this endocytosis was totally inhibited by mannose-6-phosphate. Cathepsin D endocytosis in cancer cells was not mediated by lectins or another receptor binding the cathepsin profragment. It was not due to fluid endocytosis, since another protein pS2 secreted by MCF-7 was not endocytosed with its antibody in the same conditions. Double-immunofluorescence and confocal microscopy analyses revealed that antibodies specific to pro-cathepsin D (M2E8) and to the mannose-6-phosphate/IGFII receptor were co-internalized independently in non-permeabilized MDA-MB231 cells and MCF-7 cells, but not in fibroblasts. Moreover, when metabolically labelled pro-cathepsin D secreted by MCF-7 or MDA-MB231 cells was incubated with homologous or heterologous non-radioactive cells, the time-dependent uptake and maturation of the pro-enzyme into fibroblasts were totally inhibited by mannose-6-phosphate, whereas they were not in the two breast cancer cell lines. The percentage of mannose-6-phosphate-independent binding of radioactively labelled pro-cathepsin D to MDA-MB231 cells at 16 degrees C was higher (7-8%) at low pro-cathepsin D concentration than at high concentration (1.5%), indicating the presence of saturable binding site(s) at the cell surface that are different from the mannose-6-phosphate receptors. We conclude that, in contrast to fibroblasts, breast cancer cells can endocytose the secreted pro-cathepsin D by a cell surface receptor that is different from the mannose-6-phosphate receptors or other lectins. The nature of this alternative receptor and its significance in the action of secreted pro-cathepsin D remain to be elucidated.
组织蛋白酶D的运输在癌细胞中发生改变，导致该酶原的分泌增加，该酶原可被相同的癌细胞和基质细胞重新内化。我们研究了两种人乳腺癌细胞系（MDA-MB231、MCF-7）和人正常成纤维细胞中组织蛋白酶D酶原的内吞作用。通过对活细胞内化的抗组织蛋白酶D单克隆抗体进行免疫荧光分析间接研究酶原的摄取。在存在10 mM甘露糖-6-磷酸的情况下，两种癌细胞系均将组织蛋白酶D酶原-抗体复合物内化到内体区室中。不分泌组织蛋白酶D酶原的非恶性成纤维细胞，仅在添加纯化的组织蛋白酶D酶原时才内化抗组织蛋白酶D抗体，并且这种内吞作用被甘露糖-6-磷酸完全抑制。癌细胞中组织蛋白酶D的内吞作用不是由凝集素或与组织蛋白酶原片段结合的另一种受体介导的。这不是由于液相内吞作用，因为在相同条件下，MCF-7分泌的另一种蛋白质pS2与其抗体并未被内吞。双重免疫荧光和共聚焦显微镜分析显示，在未通透的MDA-MB231细胞和MCF-7细胞中，针对组织蛋白酶D酶原（M2E8）和甘露糖-6-磷酸/胰岛素样生长因子II受体的抗体可独立共内化，但在成纤维细胞中则不然。此外，当将MCF-7或MDA-MB231细胞分泌的经代谢标记的组织蛋白酶D酶原与同源或异源非放射性细胞一起孵育时，甘露糖-6-磷酸可完全抑制该酶原在成纤维细胞中的时间依赖性摄取和成熟，而在两种乳腺癌细胞系中则不然。在16℃下，放射性标记的组织蛋白酶D酶原与MDA-MB231细胞的甘露糖-6-磷酸非依赖性结合百分比在低组织蛋白酶D酶原浓度下（7-8%）高于高浓度下（1.5%），表明细胞表面存在与甘露糖-6-磷酸受体不同的可饱和结合位点。我们得出结论，与成纤维细胞不同，乳腺癌细胞可通过一种不同于甘露糖-6-磷酸受体或其他凝集素的细胞表面受体对内化分泌的组织蛋白酶D酶原。这种替代受体的性质及其在分泌的组织蛋白酶D酶原作用中的意义仍有待阐明。
