Specific mannose-6-phosphate receptor-independent sorting of pro-cathepsin D in breast cancer cells.
The secretion of pro-cathepsin D (pro-cath-D) in some human metastatic breast cancer cells (MCF7, MDA/MB231), contrary to normal mammary cells, is not increased by ammonium chloride treatment, indicating a mannose-6-phosphate-independent sorting to lysosomes. By studying a variety of cell lines and lysosomal enzymes, we show that secretion of newly synthesized pro-cath-D was not mediated by the 46-kDa mannose-6-phosphate receptor (MPR) and that its resistance to NH4Cl for secretion was specific to cath-D and not to other lysosomal enzymes. This resistance appeared to be correlated with the basal hypersecretion of pro-cath-D, but not with its overexpression. By contrast, pro-cath-D secretion was increased by NH4Cl in fibroblasts and nontumoral epithelial mammary cells, suggesting a specificity for cancer cells. Immunofluorescence staining showed that pro-cath-D, but neither cathepsin B nor beta-hexosaminidase, accumulated in intracytoplasmic vesicles of cells treated with ammonium chloride. In pulse--chase experiments and by subcellular fractionation on Percoll gradient, cath-D was found to be sorted into dense lysosomes whether cells were treated or not by NH4Cl. Treatment of cells with NH4Cl, however, inhibited processing and maturation of pro-cath-D, which was also observed in light vesicles in the absence of NH4Cl. Part of pro-cath-D, but not processed enzyme, was also found to be membrane associated in saponin-permeabilized cells. We conclude that in breast cancer cells, the MPR-independent pathway of pro-cath-D to lysosome is predominant compared to normal cells and other lysosomal enzymes. This alternative pathway should therefore be considered, in addition to MPR, to explain pro-cath-D sorting and activation in breast cancer cells.
Institut National de la Santé et de la Recherche Médicale, Unité Hormones et Cancer (U 148), Montpellier, France.
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F
Capony
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T
Braulke
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Rougeot
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S
Roux
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P
Montcourrier
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H
Rochefort
D000643:Ammonium Chloride / Q000494:pharmacology; D000906:Antibodies; D001940:Breast; D001943:Breast Neoplasms; D002402:Cathepsin D / Q000096:biosynthesis / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000378:metabolism*; D005260:Female; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D008715:Methionine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004792:Enzyme Precursors; D017527:Receptor, IGF Type 2; D000643:Ammonium Chloride; D008715:Methionine; C058345:procathepsin D; D002402:Cathepsin D