Endocytosis of pro-cathepsin D into breast cancer cells is mostly independent of mannose-6-phosphate receptors.

Abstract

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.

• Authors: V Laurent-Matha, M R Farnoud, A Lucas, C Rougeot, M Garcia, H Rochefort
• Journal: Journal of cell science (J Cell Sci) Vol. 111 ( Pt 17) Pg. 2539-49 (Sep 1998) ISSN: 0021-9533 [Print] England
• PMID: 9701553 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies
• Enzyme Precursors
• Mannosephosphates
• Receptor, IGF Type 2
• Sulfur Radioisotopes
• mannose-6-phosphate
• procathepsin D
• Cathepsin D

Topics

• Antibodies (metabolism)
• Antibody Specificity
• Biological Transport, Active (drug effects)
• Breast Neoplasms (metabolism, pathology)
• Cathepsin D (analysis, metabolism)
• Endocytosis (physiology)
• Enzyme Precursors (analysis, metabolism)
• Female
• Fibroblasts
• Fluorescent Antibody Technique
• Humans
• Intracellular Fluid (chemistry, enzymology)
• Mannosephosphates (pharmacology)
• Protein Binding
• Receptor, IGF Type 2 (analysis, metabolism)
• Sulfur Radioisotopes
• Tumor Cells, Cultured