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Activation, processing and trafficking of extracellular heparanase by primary human fibroblasts

¹ Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel
² InSight Ltd., Rehovot, Israel
³ Weizmann Institute of Science, Rehovot, Israel
⁴ Hadassah-Hebrew University Hospital, Jerusalem, Israel

View larger version (27K): [in a new window]   Fig. 1. Acquisition of heparanase activity by primary human fibroblasts. Human foreskin fibroblasts were incubated for two hours in the absence () or the presence of 10 µg/ml recombinant 65 kDa heparanase for 30 (), 60 () and 120 () minutes. Cells (2.5x106 cells/ml) were then washed and incubated (4 hours, 37°C, pH 6.6) with sulfate-labeled ECM. Labeled degradation fragments released into the incubation medium were analyzed by gel filtration over Sepharose 6B as described in the Materials and Methods.

View larger version (90K): [in a new window]   Fig. 2. Uptake and processing of extracellular heparanase pro-enzyme. (A) Western blot analysis of cell-associated heparanase. Human foreskin fibroblasts were incubated with 10 µg/ml recombinant 65 kDa heparanase for 30, 60 and 120 minutes. At each time point, the cells were lysed and subjected to western blot analysis using anti-heparanase mAb 130. (B-D) Subcellular localization of cell-associated heparanase. Human foreskin fibroblasts were incubated with 10 µg/ml recombinant 65 kDa heparanase for 30 (B), 60 (C) and 120 (D) minutes. The cells were fixed, permeabilized and subjected to immunofluorescent staining with anti-heparanase mAb 130. Note the accumulation of heparanase in granules at 60 (C) and 120 (D) minutes. Bar, 20 µm.

View larger version (45K): [in a new window]   Fig. 3. Endosomal localization of heparanase. Human foreskin fibroblasts were incubated with 10 µg/ml recombinant 65 kDa heparanase, and labeled for endosomes (TRITC-labeled ConA) and lysosomes (LysoTracker) for 120 minutes. Following fixation and permeabilization, the cells were stained by immunofluorescence with anti-heparanase mAb 130. Note the colocalization of the endosomal staining (red) and the heparanase-containing granules (green). The overlap is represented in yellow when the images are superimposed (third panel from top) and in fluorescent ratio image analysis (bottom panel).

View larger version (19K): [in a new window]   Fig. 4. Heparanase-containing granules do not contain the EEA1 early endosomal protein. Human foreskin fibroblasts were incubated with 10 µg/ml 65 kDa heparanase for 120 minutes. The cells were then fixed, permeabilized and subjected to double immunofluorescent staining of heparanase (green, left image) and EEA1 (red, middle image). The superimposed image (right image) shows no colocalization of heparanase and EEA1. Bar, 20 µm.

View larger version (26K): [in a new window]   Fig. 5. Involvement of the actin cytoskeleton, but not microtubules, in heparanase endocytosis. Human foreskin fibroblasts were incubated with 10 µg/ml 65 kDa heparanase for 120 minutes in the absence (control) or presence of cytochalasin D or nocodazole. After fixation and permeabilization, immunofluorescent staining was performed with anti-heparanase mAb 130, followed by Cy3-conjugated goat anti-mouse antibody. Heparanase-containing granules were detected and quantified as described in the Materials and Methods. There were no granules containing heparanase in the cytochalasin D treated cells, whereas nocodazole had no apparent effect on heparanase endocytosis.

View larger version (23K): [in a new window]   Fig. 6. Cellular endocytosis of truncated versus latent heparanase. Human foreskin fibroblasts were incubated with 10 µg/ml 65 kDa heparanase (right panels) or with 10 µg/ml processed 50 kDa heparanase (left panels) for 30, 60 and 120 minutes. After fixation and permeabilization, immunofluorescent staining was performed with anti-heparanase mAb 130. Heparanase-containing granules were detected and quantified as described in the Materials and Methods. Uptake of the processed enzyme was significantly faster. The intensity of the immunoflourescent staining, size and the number of processed heparanase-containing granules reached saturation after 30 minutes.

View larger version (50K): [in a new window]   Fig. 7. Inhibitory effect of heparin on heparanase endocytosis. (A) Human foreskin fibroblasts were incubated with 10 µg/ml 65 kDa heparanase for 120 minutes without (II) or with 30 minutes pre-incubation with 0.1 µg/ml (III) or 10 µg/ml (IV) heparin. Untreated cells (I) were used as control. After fixation and permeabilization, immunofluorescence was performed with anti-heparanase mAb 130. Cells that were incubated with heparanase and heparin showed no heparanase-containing granules. (B) Intensity (in arbitrary units) and area (in µm2) of heparanase-containing granules and (C) number of granules per cell were significantly lower in cells incubated with 1 µg/ml heparin (BIII and C) compared with control cells incubated with heparanase alone (BII and C) and with untreated cells (BI and C). (D) Western blot analysis of extracellular 65 kDa heparanase binding and processing in cells incubated for 120 minutes at 37°C with 10 µg/ml heparanase alone (lane 1) or with 10 µg/ml heparanase plus 1 µg/ml heparin (lane 2) or with 10 µg/ml heparanase at 4°C (lane 3). Note that heparanase binding was only partially inhibited by heparin, and its processing was not affected by heparin. Filled arrowhead, 65 kDa heparanase precursor; empty arrowhead, 50 kDa processed heparanase.

View larger version (23K): [in a new window]   Fig. 8. Uptake, activation and trafficking of extracellular heparanase precursor. Heparanase precursor binds to the plasma membrane via HSPGs and additional putative receptor(s). The 65 kDa enzyme is processed on (or adjacent to) the cell surface, independently of HSPGs. Uptake occurs within 60 minutes, with accumulation of the processed 50 kDa form in endosomes. Heparanase endocytosis depends on the actin cytoskeleton and HSPGs, and may be inhibited by the N-terminal pro-peptide. Apparently, heparanase endocytosis and processing are two independent events. Following endocytosis, heparanase-containing granules are stored within the cell for a prolonged period. These events may control the activation, clearance and storage of extracellular heparanase.