Altered protease activity is thought to be important in tumour cell invasion and metastasis, and to have a profound role in angiogenesis. Implicated proteases belong to the serine, metallo-, aspartic and cysteine protease classes. The latter comprises more than 30 protein families [1], including family C1 with mammalian enzymes like cathepsins B and L involved in cancer growth and metastasis [2]. Since the involvement of cathepsin B in cancer metastasis was originally described by Sloane et al. [3], cathepsins, and especially cathepsin B, have been studied thoroughly. The activity of the C1 family of cysteine proteases is balanced by tight-binding inhibitors, the cystatins [4]. The cystatin protein family comprises three major groups of inhibitors: type 1 cystatins, also called stefins, which are intracellular proteins present in most cells (cystatin A and B); type 2 cystatins, which are extracellular inhibitors found in most body fluids (cystatin C, D, E ⁄ M, F, G, H, S, SA and SN); and type 3, which are multidomain proteins, the kininogens. Among the Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 lm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.Abbreviations CLSM, confocal laser scanning microscopy; DOL, degree of protein labelling; PCI, potato carboxypeptidase inhibitor.
Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin (≈ 25% of the cystatin F in 2‐ or 4‐day culture medium). By contrast, U937 cells contained only 3–4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA‐ and CAAT‐box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPα, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all‐trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong (≈ 18‐fold) down‐regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down‐regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.
Background:The secreted protease inhibitor cystatin C is internalized into cancer cells. Results: Cystatin variants with altered uptake characteristics were identified, and shown to differently inhibit intracellular cathepsin B and legumain activities. Conclusion: The internalization process, and hence intracellular enzyme activity, can be modulated by selected cystatin variants. Significance: The cystatin C uptake system may be targeted to control cancer-promoting activities of tumor cells.
Cystatin C displays the strongest inhibitory activity of all cystatins toward lysosomal cysteine proteases in general and has a widespread distribution in human tissues and body fluids, including seminal plasma. The aim of this study was to investigate the distribution of cystatin C in the male reproductive system. Immunohistochemistry revealed a widespread distribution of cystatin C in normal tissues from the testis, epididymis, vas deferens, seminal vesicle, and prostate gland. Immunoreactive cystatin C was localized in basal and secretory epithelial cells, but also in neuroendocrine cells in the prostate, identified by immunostaining for chromogranin A. On adjacent tissue sections, we demonstrated local production of cystatin C utilizing nonradioactive in situ hybridization with a 201-base-long digoxigenin-labeled antisense RNA probe specific for the cystatin C transcript. Staining patterns obtained by immunohistochemistry and in situ hybridization correlated well. Enzyme-linked immunosorbent assay for quantitative analysis of cystatin C demonstrated that cystatin C was present at high concentrations in tissue homogenates from all locations investigated, compared to liver, muscle, spleen, and other general tissues. Western blotting of tissue homogenates revealed a predominant 15-kd cystatin C immunoreactive component in accordance with previous findings in other organs. Quantitative real-time polymerase chain reaction analysis to determine messenger RNA levels in whole tissue extracts showed that the cystatin C gene is highly expressed in the seminal vesicles and the prostate gland, indicating that the major amount of cystatin C in the male reproductive organs and seminal plasma is produced by cells in these 2 tissues. It is concluded that cystatin C is highly expressed and widely distributed throughout the male genital tract, suggesting that cystatin C is an important regulator for normal and pathological proteolysis in the male reproductive system.
Cathepsin B is present in several tissues and cell types throughout the rat eye. It is localized to cytoplasmic granules, presumably lysosomes. Our results suggest that it is probably also produced in the same cell types.
The ratio between proteases and their inhibitors is unbalanced in cancer. The cysteine protease inhibitor cystatin C is internalized by some cancer cells, which affects cellular properties. Here we aimed to investigate if uptake of cystatin C and the related inhibitor cystatin E/M occur in melanoma cell lines and to evaluate to what extent the uptake affects the legumain activity that is typically increased in melanoma. First we studied the basic expression, secretion, and intracellular content of all type 2 cystatins as well as expression and activity of their possible target enzymes legumain and cathepsin B in MDA-MB-435S, A375, and C8161 melanoma cells. Legumain activity was measureable in all cell lines, and of the potential legumain inhibitors, cystatin C, E/M, and F, cystatin C was the one mainly produced. All cells internalized cystatin C added to culture media, leading to increased intracellular cystatin C levels by 120-200%. Cystatin E/M was internalized as well but at a modest rate. The effects on intracellular legumain activity were nevertheless pronounced, probably because the cells lacked this inhibitor, and its affinity for legumain is 100-fold higher than that of cystatin C. Likewise, the low-degree uptake resulted in reduced migration and invasion of A375 cells in Matrigel to an extent comparable with the W106F variant of cystatin C with optimal uptake properties and resulting in much higher intracellular levels. Thus, cystatin E/M appears to be a good candidate to efficiently down-regulate the increased legumain activity, possibly important for the malignant phenotype of melanoma cells.
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